BCEM 547 Signal Transduction and Regulation of Metabolism PDF

BCEM 547 Signal Transduction and Regulation of Metabolism Dr G Moorhead Insulin receptor Insulin Signaling, by David Goodsell Acknowledgement: Illustration by David S...

BCEM 547 Signal Transduction and Regulation of Metabolism Dr G Moorhead Insulin receptor Insulin Signaling, by David Goodsell Acknowledgement: Illustration by David S. Goodsell, RCSB Protein Data Bank. doi: 10.2210/rcsb_pdb/goodsell-gallery-014 It’s crowded in here 2 Nature, 2021  BCEM 547 Signal Transduction and Regulation of Metabolism What is Signal Transduction? Signal transduction refers to the biochemical processes by which cells respond to cues in their internal or external environment. It is interesting to note that the top four classes of drug targets today play a role in signaling: G-protein coupled receptors (GPCRs), protein kinases, proteases and ion channels. To survive, cells must communicate with their neighbours, monitor environmental conditions & respond appropriately to a host of different stimuli that impinge on their surface. Cells must also monitor intracellular changes, perturbations, damage and resource/nutrient levels. In all cases some signal must be …….... or interpreted, and an appropriate ……...……... initiated. Cell signaling makes it possible for cells to talk to each other & for an organism to function as a coherent system. Cell signaling is also intimately involved in regulation of cell growth & division, making it important in understanding the development of a malignant tumor. Signal Transduction- the basics 3 Altered protein synthesis The Basic Elements of Cell Signaling Systems I. Cells usually communicate with each other through extracellular messenger molecules – cell signaling starts with release of a messenger molecule by a cell engaged in sending messages to other cells in body. The messenger molecule may have to circulate throughout the entire body before reaching specific target cells-the endocrine system- i.e. hormones. Often intracellular derived signals are generated and responded to. For instance, if DNA is damaged, the cell must recognize this, initiate repair and coordinate this with other events in the cell, such as the cell cycle. Cells must also tightly monitor metabolites and nutrients and ‘decide’ to grow or not and this must be linked to information from hormones such as insulin. II. Cells respond to an extracellular message if they express ……...……...…………..…... that specifically recognize & bind that particular messenger molecule. Binding of the messenger molecule (ligand) to the extracellular surface of the Each cell type receptor relays a signal across the expresses membrane to the receptor's cytoplasmic specific domain at the inner membrane surface. receptors, ex.? PM enzyme recruiting platform III. Once it has reached the inner plasma membrane surface, there are 2 major routes by which the signal is transmitted into the cell interior (with 2 minor exceptions: Ligand gated channels Steroid hormone receptors Cellular Response 4 From Karp, Cell and Molecular Biology: Concepts and Experiments, 4th Edition, Wiley Routes  One type of receptor transmits signal from its cytoplasmic domain to a I nearby enzyme that generates a ……..……....……... o Second messengers are small substances that typically activate (or inactivate) specific proteins. o Depending on its chemical structure, a second messenger may diffuse through the cytosol (IP3) or remain embedded in the membrane lipid bilayer (PI-3,4,5- P3).  Another type of receptor transmits a signal by transforming its II cytoplasmic domain into a ……..……....……... for cellular signaling proteins. IV. Whether the signal is transmitted by a second messenger or by protein recruitment, the outcome is similar, a protein that is positioned at the top of an intracellular signaling pathway is activated. Each signaling pathway consists of a series of distinct proteins that operate in sequence. Each protein in the pathway typically acts by altering the conformation of the subsequent (downstream) protein in the series, an event that activates or inhibits the protein- this is a ……..……....………... Most often this switch is ‘flipped’ by a ……..……........……... of the target protein (see below). Signals transmitted along such signaling pathways ultimately reach target proteins involved in basic cell processes; depending on cell type & message, the response initiated by the target protein may involve: 1. A change in gene expression 2. A change in protein synthesis 3. An alteration of the activity of metabolic enzymes 4. A reconfiguration of the cytoskeleton 5. An increase or decrease in cell mobility V. Finally, signaling has to be terminated so that cells canbe responsive to additional messages that they may receive. 5  A tangent on proteins and covalent modifications Concept of Proteoforms - the term ‘proteoform’ is used to designate all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations (SNPs), non-conventional translation, proteolysis, alternatively spliced RNA transcripts, unrecognized ORFs in main ORF, ORFs within lncRNA and post-translational modifications. This concept will be important at the end of Part I when we try to resolve specificity in signal transduction. Alternate start site Gene Product H From Smith et al Nature Methods 10, 187-88 Note: the additions of unrecognized ORFs in main ORF, ORFs within lncRNA above stem from a 2020 paper (Chen et al. Science (2020) 367 (6482), 1140-1146) that identified hundreds of previously uncharacterized functional micropeptides in the human genome….see next pg...this is re-writing the textbooks material! 6  Expanding the human proteome: functional translation of noncanonical human open reading frames Using mass spectrometry, ribosome profiling, and several CRISPR-based screens, Chen et al. (Science (2020) 367 (6482), 1140-1146) identified hundreds of previously uncharacterized functional micropeptides in the human genome. Protein translation outside of annotated open reading frames (ORFs) in messenger RNAs and within ORFs in long noncoding RNAs is pervasive. Micropeptides encoded by multiple short, upstream ORFs often form stable protein complexes with the downstream canonical proteins encoded on the same messenger RNAs. unrecognized ORF an operon? 2025 update: 7264 noncanonical ORFs in the human genome and to date ~3000 of these mini-proteins have been found.  Post-translational Modification of Proteins. Why have covalent modifications of amino acid side-chains? The 20 amino acids constrain the functional and structural repertoire of proteins. By adding modifications this makes an almost limitless set of altered ‘……..……....……...’ The cellular response to changing conditions is frequently mediated by the reversible covalent modification of existing molecules which also often allows easily reversible modification and thus function- or it has the potential to create a molecular switch. Fast: to add or remove a modification, slow to make a protein. Cheap: to add or remove a modification, expensive (E and a.a.) to keep making a protein. ?? The prototype in eukaryotic cells is protein …………...……....……... of the hydroxy- amino acids Tyr, Ser and Thr. However, proteins can be altered by a diverse set of post-translational modifications (PTMs), including the methylation of Arg residues, the methylation, acetylation (acylation), ubiquitylation or sumoylation of Lys residues, oxidation or lipidation, among many others… 8 © 2023 The Author(s). This is an open access article published by Portland Press Limited on behalf 1242 of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Glycoproteins are proteins to which one or more oligosaccharides have been attached through covalent bonding. Orange is covalently attached carbohydrate or glycan SARS-CoV-2 virus that causes COVID-19 (dark blue) is covered in spike proteins (light blue), which are glycoproteins that allow the virus to gain access to human cells. 9  Post-translational Modification of Proteins. It is thought that protein phosphorylation is the most common covalent modification, with ubiquitylation and acetylation ranking 2nd and 3rd. An individual polypeptide chain can potentially be phosphorylated at several sites, thereby producing many phospho-isoforms, each of which can potentially have a distinct biological activity. Furthermore, the same polypeptide chain can be modified by different classes of PTMs, which generates an even larger number of possible variants. N P N C P P P C P P Ac P Me What can covalent modification of proteins do? 1. alter protein ……..……....…... in the cell. 2. alter ……..……....……... 3. control ……..……....……... 4. provide a specific ……..……....……... for other proteins or molecules. 10 Quick review: the adenylates AMP or 5’-AMP ADP ATP 11 Protein Kinase/Phosphatase Cycles Act as Molecular Switches Unlike in an allosteric effector system, protein phosphorylation and dephosphorylation are brought about by different reactions: A PROTEIN KINASE/PHOSPHATASE CYCLE: Phosphoryl group ATP or Mg-ATP? transfer Mg- History of Protein Phosphorylation Intimate to the study of signal transduction and biochemical regulation, is protein phosphorylation. It is now known that 7 in 10 proteins in mammalian cells are phosphorylated in their lifetime. Protein kinases and protein phosphatases catalyze phosphorylation/ dephosphorylation reactions, respectively. Genome sequencing has revealed that about 3% of all human genes encode protein kinases (~518 genes) and phosphatases (~147). The realization that protein phosphorylation/ dephosphorylation constitutes an important regulatory mechanism was slow in developing, unlike the sudden and universal acceptance of allosteric regulation proposed by Monod and colleagues in the mid-1960s. Critical to the development of this field were studies on glycogen metabolism. -in the 1940s Carl and Gerti Cori showed that glycogen phosphorylase existed in 2 forms they called phosphorylase b and phosphorylase a. Phosphorylase b was active only in the presence of high concentrations of ……….….., whereas phosphorylase a was highly active in the absence of 5’-AMP. They showed the 2 forms were interconvertible in cells, but it was another 10 years before Earl Sutherland’s lab and Fischer and Krebs demonstrated that this was due to phosphorylation/ dephosphorylation. 12  Carl and Gerti Cori- Nobel Prize for studies on glycogen metabolism Fischer and Krebs were awarded the Nobel Prize in Medicine in 1992 for their work. Sandwalk: Nobel Laureates: Edmond Fischer and Edwin Krebs Phosphorylase kinase P P Phosphorylase phosphatase Phosphorylase b Phosphorylase a (inactive) (active) in 1959, Ed Krebs was able to show that phosphorylase kinase was itself regulated by phosphorylation. Is this a biochemical mechanism restricted to a limited area of carbohydrate metabolism? 13 The phosphorylation of proteins occurs on 5 residues: ? % (percentages) refer to the human cell phospho-proteome based on quantitative mass spectrometry. 1) phosphoserine (86%) 2) phosphotyrosine (1.8%)   3) phosphothreonine (12%) See PubChem  Now found in Eukaryotes The formation of phosphohistidine and aspartyl phosphate is only known to occur in two-component systems. 4) phosphohistidine 5) aspartyl phosphate 14 Covalent modifications often act as docking sites for domains or modules in other proteins SH2 N C Domains or modules are within a protein sequence within a protein sequence P-Tyr Squiggly lines SH2 mean ‘within a protein’ Me-Lys chromodomain Ac-Lys Note charge loss bromodomain /KAT /KDAC ubiquitin UIM Ubiquitin interaction module From Seet et al 2006. NATURE REVIEWS | MOLECULAR 15 CELL BIOLOGY p473-83.  Concept of domains and modular organization ofproteins. Domains and modules: where and why? proteins are constructed in a modular fashion from a combination of interaction, structural and catalytic domains. N C The pathways and networks that link receptors to their ultimate targets frequently involve a series of protein-protein interactions, that recruit and confine signaling proteins to an appropriate subcellular location and determine the specificity with which enzymes interact with their targets. How do we define a protein motif, domain or module? A motif is typically a 3-10 a.a. sequence and is sometimes referred to as a SLiM [Short Linear Motif]. Typically, protein-protein interaction domains are independently folding modules of ………….………………, which can be expressed in isolation from their host proteins while retaining their intrinsic ability to bind their physiological partners. ligand binding Their N- and C-termini are usually close together in space, site while their ligand-binding surface lies on the opposite face of the domain. This arrangement allows the domain to be inserted into a new protein (gene) while projecting its ligand-binding site to engage another polypeptide (protein evolution). Q: w.r.t. domains and modules, what does the human genome tell us about protein evolution? 16  Families of interaction domains Protein-protein interaction domains can be divided into separate families, that are related either by sequence or ligand-binding properties. Src Homology 2 P-tyrosine binding (SH2 and PTB) A large number of cytoplasmic proteins contain one or two SH2 domains that directly recognize ….……….…..…-containing motifs, such as those found on activated receptors for growth factors (RTKs). The surrounding a.a are important too! X-X-X-pY-X-X-X SH2 domains were initially identified as part of protein-tyrosine kinases encoded by the genome of tumor-causing (oncogenic) viruses. Peyton Tony Rous 1910 Rous Sarcoma virus…Src protein kinase (1st viral oncogene) Pawson 1980s Interaction domains are often used repeatedly in numerous different proteins to mediate a particular type of molecular recognition, and indeed the human genome is predicted to encode 120 SH2 domains. However, phosphotyrosine-containing motifs are also recognized by a quite different class of interaction modules, termed PTB (phosphotyrosine binding) domains, found on docking proteins such as the IRS- 1 substrate of the insulin receptor. From Karp, Cell and Molecular Biology: Concepts and Experiments, 4th Edition, Wiley SH2 drives protein: protein interactions N and C-termini 17 How does the Rous sarcoma virus cause cancer? Src PK Peyton Rous 1910- the actual chicken where the tumor was taken from, ground up, cells filtered out and the soluble portion used to induce tumor growth in other chickens. 18 P-serine/threonine binding A large family of interactions domains, including 14-3-3 proteins, FHA and MH2 domains and BRCT domains recognize specific phosphoserine/threonine motifs, and thereby mediate the biological activities of protein-serine/threonine kinases. -FHA domains bind pT-containing SLiMs X-X-X-pT-X-X-X -BRCT domains bind pS-containing SLiMs X-X-X-pS-X-X-X Acetyl- and methyl-lysine binding Acetylation or methylation of lysine residues on histones creates binding sites for the ………..……… and …………….. domains, respectively, of proteins involved in chromatin remodeling. Although discovered on histones, we now know 1000s of proteins are acetylated on K Modular organization = string domains/modules together to create a new protein Different interaction domains are frequently found within the same polypeptide chain, to yield a protein that can mediate multiple protein-protein, protein-phospholipid, protein- carbohydrate and protein-nucleic acid interactions. 19 Examples of modular proteins binds poly-proline Growth factor receptor-bound protein 2 SH1 GTPase Activating Protein for Ras ? ? From http://pawsonlab.mshri.on.ca/images Protein and Phosphoinositide Interaction Domains… 14-3-3 ADF ANK ARM BAR BEACH BH14 BIR BRCT BROMO BTB C1 C2 CARD CC CH CHROMO CUE DD DED DEP DH EFH EH ENTH EVH1 FBOX FERM FF FH2 FHA FYVE GAT GEL GRAM GRIP GYF HEAT HECT IQ LIM LRR MH1 MH2 PAS PB1 PDZ PH POLO_BOX PTB PUF PX RGS RING SAM SCD SH2 SH3 SOCS START TIR TPR TRAF TSNARE TUBBY TUDOR UBA UIM VHLB VHS W WW… The Encyclopedia of Domains-Lau et al., Science 386, 508 (2024) AlphaFold Protein Structure Database contains predictions for more than 214 million protein structures…. TED identifies nearly 365 million domains across more than more 20 than 1 million taxa.  The Concept of Reader, Writer and Eraser Knowing that specific domains can recognize and dock covalent modifications in a sequence specific context, the idea emerged that reversible covalent modifications function like a ………..……… to send information where one enzyme writes, one enzyme erases and a domain within another protein reads the message. From- Pincus, David et al. (2008) Proc. Natl. Acad. Sci. USA 105,9680-9684 by protein: protein interactions X-X- -X-X This idea can be extended to other enzyme pairs and domains, like HATs, HDACs and bromodomains. It can also be extended to other molecules, like phosphoinositides and their specific docking partners (ex. PH domains that bind PI-3,4,5-P3) [coming soon!]. Note HATs and HDACs now renamed to KATs and KDACs. 21  Back to general signaling concepts… A Survey of Extracellular Messengers and Their Receptors I. A large variety of molecules can function as extracellular carriers of information, including: Small molecules like amino acids and amino acid derivatives – glutamate, glycine, acetylcholine, epinephrine, dopamine & thyroid hormone; these molecules act as neurotransmitters & hormones. Gases eg. NO (nitric oxide). Steroids, which are derived from cholesterol. Eicosanoids. II. Extracellular signaling molecules are usually, but not always, recognized by specific receptors that are present on the surface of the responding cell. The receptors that have evolved to mediate signal transduction are: 2 major routes ……………..…………………………………….… (GPCRs) – huge family of receptors that contain 7 transmembrane -helices. They translate binding of extracellular signaling molecules into activation of GTP-binding proteins. ………………………………………………….…………… (RTKs) – a second class of receptors that have evolved to translate the presence of extracellular messenger molecules into changes inside the cell. P RTK o Binding of a specific extracellular ligand to RTK results in activation of the receptor's protein-kinase activity, which is associated with its cytoplasmic domain. o Upon activation, these protein kinases autophosphorylate to create a docking platform. Note, some Ser/Thr receptor kinases do exist, eg. TGF receptor (slide 89) 22  The receptors that have evolved to mediate signal transduction are: Ligand-gated channels – cell surface receptors that bind to extracellular 2 minor routes ligands; their ability to conduct a flow of ions across the plasma membrane is regulated directly by ligand binding. Steroid hormone receptors- function as ligand-regulated transcription factors (T.F) o Steroid hormones diffuse across plasma membrane & bind to their receptors, which are present in the cytoplasm. o Hormone binding results in conformational change that causes hormone- receptor complex to move into the nucleus & bind to promoters or enhancers of hormone responsive genes. o This interaction gives rise to an increase or decrease in the rate of gene transcription. Free DNA Cliparts, Download Free Clip Art, Free Clip Art on Clipart Library Steroid hormone T.F. P.M. G Protein-Coupled Receptors: Background Information on Receptors G-protein coupled receptors (GPCRs) – are so-named because they interact with G proteins; also referred to as ………………………………………………….…… because they contain 7 transmembrane helices. o Hundreds of different GPCRs have been identified in organisms ranging from Only 2 yeast to plants & mammals; they regulate an extraordinary spectrum of cellular processes. o They constitute the single largest protein ……………………………………… encoded by animal genomes and are the number one drug target of the pharmaceutical industry! 959 cells Worms: Models of Development o The nematode C. elegans whose genome contains 19,000 genes, encodes ~1000 different GPCRs, while humans have ~800 with (in)activating mutations linked to >30 human diseases! So many o Included among the natural ligands that bind to GPCRs are a diverse array of hormones, chemoattractants, neurotransmitters, opium ligands derivatives, odorants, tastants & photons. 23 Included among the natural ligands that bind to GPCRs are a diverse array of …….odorants. In vertebrates, the vast majority of odorants are detected by odorant receptors (ORs), which are G protein-coupled receptors (GPCRs) expressed in olfactory sensory neurons. 400 ORs propionate. cAMP Fig. 1 | Structure of the human OR OR51E2. a, Phylogenetic tree of human class A GPCRs, including both non-olfactory and ORs. b, Real-time monitoring of a cAMP concentration assay showing that human OR51E2 responds to the odorant propionate (a fatty acid). c, d, Cryo-EM density map (c) and ribbon model (d) of active human OR51E2 bound to propionate (yellow spheres). OR51E2 is bound to Gαs and bound to both Gβγ and the stabilizing nanobody Nb35. 24 Nature. 2023 Mar;615(7953):742-749  Ligand binding to GPCRs Odorant-binding pocket in OR51E2 (a) is similar to the prototypical class A GPCRs: β2AR bound to adrenaline and (b) and rhodopsin bound to all-trans retinal (c). TM, transmembrane a-helix. 25 G protein-coupled receptors normally have the following topology: outside cell P.M.= plasma membrane Inner leaflet of the plasma membrane From Karp, Cell and Molecular Biology: Concepts and Experiments, 4th Edition, Wiley i.e. a GPCR Synthesize G peptide G N C  NATURE|Vol 455|25 September 2008 Figure 1 | Activation of a G-protein-coupled receptor. a, Rhodopsin, shown here in its inactivated conformation, is a light-sensing receptor found in cell membranes. It consists of a protein (opsin, green) and a ligand (retinal, pink, also shown in its inactivated conformation). When activated by light, rhodopsin binds to part of an adjacent G protein (binding region in red), triggering a cascade of biological responses. The protein plug (blue) is part of the extracellular domain of opsin and immobilizes the extracellular transmembrane segments of the receptor. Scheerer et al. have determined the activated structure of opsin in complex with the receptor-binding peptide fragment of the G protein (the G peptide). The most notable difference when compared with the inactivated receptor is that transmembrane helix 6 (TM-VI) has moved substantially outward (indicated by the red arrow), thereby 26 creating the binding pocket for the G-protein peptide. Function and structure of the Adenylate Cyclase9-G complex. effector G Active site: ATP → cAMP Chao Qi et al. Science 2019;364:389-394 Cryo-EM density map of the AC9-Gαs complex allowed us to build the complete molecular model of the complex (E), revealing its key structural elements: the transmembrane domain bundle (TM), the catalytic domain of AC9 (C2a), and the G protein. The density map and model elements corresponding to AC9 and the Gαs proteins are colored orange and green, respectively. Detergent and unassigned protein density are colored gray. Adenylate cyclase (AC) catalyzes the synthesis of cAMP from ATP. Protein-Coupled Receptors: G Proteins General Structure and Function I. Heterotrimeric G proteins were discovered & characterized by Martin Rodbell et al at the NIH in the early 1970s – they are called G proteins because they bind guanine nucleotides, either GDP or GTP. A. They are described as ………..heterotrimeric…… because all of them consist of 3 different polypeptide subunits (  and ), distinguishing them from small, monomeric G proteins, like Ras. B. Heterotrimeric G proteins are held at the plasma membrane by lipid chains that are covalently attached to the  &  subunits. C. The guanine-nucleotide-binding site is present on the G  subunit. 27 GDP or GTP  I. When a hormone or neurotransmitter binds to a GPCR, it induces a change in conformation in the extracellular ligand-binding site. o The change in conformation is transferred across the plasma membrane & causes a change in conformation in the cytoplasmic loops inactive of the receptor. o This, in turn, leads to an increase in the receptor's affinity for a G protein that Adenylate cyclase is present on the cytoplasmic surface —> the ligand-bound receptor forms a receptor-G protein complex. o The interaction with the receptor induces a conformational shift in the  subunit of the G protein, causing the release of GDP, which is followed by the binding of GTP —> G protein is activated. While in the activated state, a single receptor can activate a number of G protein molecules, providing a means of signal amplification. GRK From Karp, Cell and Molecular Biology: 28 Concepts and Experiments, 4th Edition, Wiley II.Replacement of GDP by GTP after an interaction with an activated GPCR causes a conformational change in the G subunit. o In its GTP-bound conformation, the G subunit has a low affinity for G, leading to its dissociation from the complex. Each dissociated G subunit with GTP attached is free to activate an effector protein like adenylyl cyclase, which in this case leads to the production of the second messenger, cAMP. o Second messengers, in turn, activate one or more cellular signaling proteins. o After its interaction with an effector, G hydrolyzes its bound GTP to GDP and Pi. o GTP hydrolysis induces a shape change causing G subunit to dissociate from effector & to reassociate with  dimer forming an inactive heterotrimeric G protein, waiting for next round of activation. What about the  complex III. After its dissociation from the G subunit, the  complex also has a signaling function & it can couple to at least 3 different types of effectors: PLC, K+ ion channels & PI 3-kinase. All ‘associated’ with the plasma membrane …does this make sense? 29  G Protein-Coupled Receptors: G Proteins -Response Termination Step 1 of desensitization – the cytoplasmic domain of the activated receptor (GPCR) is phosphorylated by a specific type of kinase, G protein-coupled receptor kinase (GRK). The conformational changes that make it possible for GPCRs to activate G proteins also make them good GRK substrates; thus, after ligand binding, GPCRs become sensitive to GRK phosphorylation (molecular switch). Step 2 – GPCR phosphorylation sets the stage for the second step, which is the binding of proteins (arrestins). Arrestins form a small group of proteins that bind GPCRs & compete for binding with heterotrimeric G proteins —> thus, arrestin binding prevents further activation of additional G proteins. Signaling by the activated G subunit is terminated by a very different mechanism: the bound GTP molecule is simply ………………………………….. Thus, strength & duration of the signal are determined partly by the G subunit GTP hydrolysis rate. Once the GTP is hydrolyzed, the G-GDP reassociates with the G subunits to reform the inactive trimeric complex —> returns system to the resting state. 30 G Protein-Coupled Receptors: Ancient Proteins and Bacterial Targets I. Mechanism for transmitting signals across the plasma membrane by G proteins is of ancient evolutionary origin & is highly conserved. Bacterial toxins – because G proteins are so important to the normal physiology of multicellular organisms, they have been targeted by bacterial pathogens. o Cholera toxin (produced by Vibrio cholerae) exerts its effect by modifying G-subunits & inhibiting their … ……………………………………………… in the cells of the intestinal epithelium. o Adenylyl cyclase molecules remain in the activated mode, churning out cAMP, which causes the epithelial cells to secrete large volumes of fluid into the intestinal lumen. o The loss of water associated with this often leads to death due to dehydration. o Pertussis toxin is one of several virulence factors produced by Bordetella pertussis, a microorganism that causes whooping cough. o Pertussis toxin also acts by ………………………………………………… G-subunits, which interferes with the signaling pathway that leads the host to mount a defensive response against the bacterial infection. 31  The h u m a n ge nome enc odes a s ma ny a s 8 00 different GPCRs. They are very i mportant, since > 1 / 3 of all …………………………… act as ligands that bind to this hug e superfami ly of receptors. Second Messengers The Discovery of a Second Messenger – Cyclic AMP [cAMP] The discovery of a 2nd messenger: cyclic AMP – mid-1950s; Earl Sutherland et al. and Edwin Krebs & Edmond Fischer show how a hormone alters cytoplasmic enzyme activity. o They want to determine physiological responses to hormone; studied with in vitro system of broken cells. Eventually, he activated glycogen phosphorylase in a broken- cell preparation (crude extract) exposed to glucagon or epinephrine [adrenalin]. Then they divided the broken-cell preparation into particulate fractions (mostly cell membranes) & soluble fractions by centrifugation. Crude extract or spin or Soluble (phosphorylase) broken-cell Particulate (membranes) preparation Add hormone and phosphorylase is activated Add hormone and phosphorylase is NOT 32 activated  ▪ Phosphorylase was present only in supernatant (soluble fraction), but the response to hormone required the particulate fraction – experiments show that it is at least a 2-step response. Isolate particulate fraction, treat with hormone, wash & add wash to supernatant —> phosphorylase activated; wash contained 'substance' that activated phosphorylase. Treat with hormone, spin and add supernatant back to Resuspend soluble fr.= activated pellet phosphorylase phosphorylase 1. Sutherland identified the substance released by the particulate fraction membranes as cyclic adenosine monophosphate (cyclic AMP or cAMP). i.e, …………………. 2. cAMP —> glucose mobilization stimulated; activates a protein kinase that adds a phosphate group to a specific serine residue of the phosphorylase polypeptide —> activates phosphorylase. cAMP -> PKA -> PhK -> Phos b -> Phos a = glycogen degradation (see slide 12+13) cAMP is 2nd messenger - released into cytoplasm due to 1st messenger (hormone or other ligand) binding at cell outer surface & diffuses to other sites in cell. PKA= Protein Kinase A Second Messengers 1. While ………………………………………… bind only to a single receptor species, ……………………………… stimulate a variety of cell activities leading to large- scale, coordinated response after stimulation by single extracellular ligand. For ex. PKA has many substrates 2. A number of other 2nd messengers have been found in eukaryotic cells: Phosphoinositides, inositol triphosphate, diacylglycerol, Ca2+ ions, cGMP. Blue= lipid derived 33  Lipid-Derived Second Messengers – Background I. Cell membrane phospholipids were originally viewed as structural components that make membranes cohesive & impermeable to aqueous solutes. PLASMA MEMBRANE STRUCTURE AND FUNCTION TRANSPORT ACROSS MEMBRANE. - ppt download of plasma membrane Relative levels of acidic or negatively charged phospholipids in mammalian cells compared to total lipids With a growth factor Seven EGF NGF phospho- PDGF. inositides PI-3,4,5P3 low changes 34  Cell culture and growth factors Red = media with nutrients, bovine serum Appendix: Growth Factors  When investigators began studying the effects of biological substances on cells and tissues in culture, they discovered a group of peptide-hormone-like substances that wer e distinct from any previously known hormones (i.e. growth factors).  A growth factor is a naturally occurring substance capable of stimulating cellular growth, ? proliferation, healing, and cellular differentiation. Usually, it is a protein or a steroid hormone.  Growth factors typically act as signaling molecules between cells. Examples are cytokines and hor mones that bind to specific receptor s on the surface of their target cells.  Examples: epidermal growth factor [EGF], which stimulates the growth of epithelial cells; platelet-derived growth factor [PDGF], which stimulates the growth of muscle cells and connective tissue cells; and nerve growth factor [NGF] , which stimulates the growth of neuronal cells. A. It is now known that phospholipids form precursors of a number of ………………………………………phospholipids are converted into 2nd messengers by a variety of enzymes that are regulated in response to extracellular signals. B. The enzymes that convert phospholipids into 2nd messengers include: Phospholipases (lipid-splitting enzymes) Phospholipid kinases (lipid-phosphorylating enzymes) Phospholipid phosphatases (lipid-dephosphorylating enzymes) PTEN C. Phospholipases are enzymes that hydrolyze specific ester bonds that connect the different building blocks that make up a phospholipid molecule; there are 4 classes of these enzymes (PLA1, A2, C and D). They are activated in response to extracellular signals The products they make function as second messengers 35  PLC A generalized phospholipid; the PH domain of a PLC docking a membrane through a phosphoinositide. From Karp, Cell and Molecular Biology: Concepts and Experiments, 4th Edition, Wiley Best-studied lipid 2nd messengers are derived from …phosphatidylinositol. & are generated after transmission of signals by G protein-coupled receptors and RTKs. Lipid-Derived Second Messengers – Phosphatidylinositol (PI)-Derived Second Messengers First indication that phospholipids might be involved in cell responses to extracellular signals emerged from studies below. 1st messenger Lowell & Mabel Hokin -early 1950s- they had set out to study acetylcholine effects on pancreatic ………………………………………………….… Incubate pigeon pancreas slices in 32PO4 (orthophosphate); this is the usual way of getting radiolabeled nucleotide triphosphates, which are used as precursors during RNA synthesis. Tissue treatment with acetylcholine led to radiolabel incorporation into cell phospholipid fraction (mostly PI); it was then quickly changed to other phosphorylated derivatives (……………………………………...) Suggested that lipids can be phosphorylated by specific lipid kinases that are 36 activated in response to extracellular messenger molecules like acetylcholine.  II. Reactions of PI metabolism – the inositol ring resides at the inner polar surface of the bilayer & has 6 carbons. 18:1 PI(4,5)P2 | Avanti Polar Lipids PI-4,5-P2 Lipid kinases outside PM ATP ATP  specific for PLC Slide 29 [1X] From Karp, Cell and Molecular Biology: Concepts and Experiments, 4th Edition, Wiley [10,000X] A. Carbon #1 is involved in joining inositol to diacylglycerol; the others can be phosphorylated by various phosphoinositide kinases present in cells. Reversible phosphorylation of its inositol ring at positions 3, 4 and 5 results in the generation of ……………………… phosphoinositide species (see below). B. The PI 3-kinases (PI3Ks), PI 4-kinases (PI4Ks) & PI 5-kinases (PI5Ks), transfer a phosphoryl group from ATP to the 3, 4 or 5 position on the inositol ring, respectively. Phospholipid binding 13 PLC isoforms docks PI-4,5-P2 in humans PLC  PH EF Cat Cat C2 37  PIP3 PKC X lipid phosphatase PI metabolism. The structures and interconversion reactions are shown for all phosphoinositides that are found in mammalian cells. The phosphoinositide kinases that catalyze the addition of phosphate groups to the 3-, 4- and/or 5-positions are shown, as are the lipid phosphatases PTEN (phosphatase and tensin homologue on chromosome 10) and SHIP (SH2-containing inositol 5'-phosphatase). The phospholipid-binding domains that recognize specific phosphoinositides and diacylglycerol (DAG) are shown. Only pleckstrin homology (PH) domains recognize PtdIns(4,5)P2, PtdIns(3,4,5)P3 or PtdIns(3,4)P2 with high specificity. All FYVE domains bind PtdIns3P, as do nearly all Phox-homology (PX) domains. Note: All of these phospholipid species remain in a cytoplasmic leaflet of a membrane. 38  How FYVE and PX domains bind PI-3-P Inside cell Inside cell Schematic Drawing (left) and Molecular Surfaces (right) of EEA1-FYVE (A) and p40phox- PX (B) Domains The molecular surfaces are shown in the same orientation as the schematic drawings and are colored blue (basic), red (acidic), green (hydrophobic), and white (uncharged polar). The structures are docked to a membrane, with the polar interface and hydrophobic core regions of the membrane shown to scale. Residues predicted to be involved in nonspecific hydrophobic and electrostatic interactions with membrane lipids other than PI(3)P are shown. 39 Cell Volume 107 Issue 5 Pages 559-562  PI3K (lipid kinases) PI3Ks are a family of enzymes that were originally defined on the basis that they could catalyze the phosphorylation of the 3-position of the ………………….... in one or more phosphoinositide substrates. Class I enzymes preferentially phosphorylate PtdIns(4,5)P2 and hence synthesize PtdIns(3,4,5)P3. Thus, Class I PI3K-generated PtdIns(3,4,5)P3 represents a major signal transduction pathway downstream of cell-surface receptors. Class1A via RTKs; Class 1B via GPCRs. There are 4 Class I PI3K genes in humans:     Routes 1 and 2 Class III PI3K-generated PtdIns3P represents a major regulatory pathway defining and controlling the passage of proteins and membranes through the endosomal/lysosomal compartments. Ras P-lipid Binding binding Domain domain PI3K PI45P2 VIA ATP RTKs VIA GPCRs See slide 27 p84/ ?? PI3K family of human enzymes. 40  Activation of Class I PI3K by Growth Factor Receptors Frontiers | The Ras Superfamily of Small GTPases in Non-neoplastic Cerebral Diseases | Frontiers in Molecular Neuroscience Small G-protein lipid = binds R R target proteins T T K K YxN SH2 YxN Pro-rich p85 PIP3 GEF= guanine nucleotide exchange factor. p85 p110 SOS is the GEF for Ras GPCR Direct activation by  Slide 27 ☺ p110 PIP3 4p110 Pp101/84 41 Activation of Class I PI3Ks by growth factor receptors (A)Activation of Class IA PI3Ks by receptors utilizing protein tyrosine kinases as their proximal signal transduction element. A schematic representation of the activation of Class IA heterodimers by a growth factor receptor with intrinsic protein tyrosine kinase activity is shown. The key features are growth-factor-induced dimerization of the receptor, transphosphorylation (-------) of multiple key tyrosine residues in the cytoplasmic tails, which act as docking sites for multiple SH2-domain- containing signalling proteins. Some of these phosphotyrosine residues are within pYXN motifs that act as docking sites for the SH2 domains in the p85 regulatory subunits of Class IA PI3Ks, leading to their recruitment into these growth factor receptor signalling complexes. GTP-Ras is known to bind to the catalytic subunit of Class I PI3Ks and is thought to play a role in the synergistic activation of their lipid kinase activity. Ras is converted into the GTP bound form by GEFs (- - - -), which are often recruited in parallel to PI3Ks by distinct phosphotyrosine residues in these signalling complexes (in the example shown, the Ras GEF, SOS, is recruited indirectly via the adaptor GRB2). (A)Activation of Class IB PI3K by receptors utilizing heterotrimeric G-proteins as their proximal signal transduction element. A schematic representation of the activation of a heterodimer of either p101 or p84 regulatory subunits and the p110γ catalytic subunit by a G-protein coupled receptor is shown. Key features are the direct binding and activation of the Class IB PI3K by Gβγ s that are liberated by dissociation of the heterotrimeric G-protein. GTP-Ras is known to bind directly to p110γ, and recent evidence indicates this interaction plays an important role in activation of the PI3K activity downstream of G- protein - coupled receptors; the mechanism by which guanine nucleotide exchange on Ras is stimulated in these contexts of receptor activation is, however, unknown. 42  Small molecule or drugs RTK G 43 Oncogenic mutations in PI3Ks PI3K is 1 of the 2 most frequently mutated ………………………………. of human cancer(s). PI3K was linked to cancer when found in the genome of avian sarcoma virus 16 (1997). ATP + PI45P2 → ………………. PM P p110 RTK ‘inner SH2’ binds p110 = ?? From Huang et al 2007 Science 318:1744-48. K and R ?? = ?? Model of membrane interaction.(A) Positively charged residues on the surface of iSH2 domain of p85 (red) and loops of the C2 and kinase domains of p110 are proposed to contact the negatively charged phospholipid bilayer. (B) Model of p110 /niSH2 bound to Ras and its proposed orientation with respect to the lipid membrane. Mutations found in cancers map to the p85 and p110 interaction residues and the kinase domain. 44 = increased PIP3 Case Study: A Case for Exome Sequencing Angulo et al. Science 15 November 2013: Vol. 342 no. 6160 pp. 866-871 Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage  expressed in immune and endothelial cells Abstract Here, we describe activated PI3K-δ syndrome, a primary immunodeficiency associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3- kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. PI3K-δ syndrome was characterized by recurrent respiratory infections, progressive airway damage, increased circulating transitional B cells, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate. Selective p110δ inhibitors IC87114 and GS- 1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with PI3K-δ syndrome. Families with the E1021K p110δ mutation. We used exome sequencing to search for causative mutations in 35 PID patients from the United Kingdom who suffered recurrent infections and had a family history of susceptibility to infections. After identification of genetic variants in these patients, we excluded common polymorphisms previously detected in the 1000 Genomes and National Heart, Lung, and Blood Institute (NHLBI) projects. When cross-checking the remaining rare variants, we noted that three patients from one family (P1, P2, and P3 in family A) and one patient from another family (P5 in family B) had the same heterozygous G to A mutation at position 9,787,030 on chromosome 1, c.3061G>A in the PIK3CD gene (Fig. 1). This mutation was not present in the other exomes and was the only rare variant shared among all patients in these two unrelated families. It encodes an amino-acid substitution, a glutamic acid for a lysine, at position 1021 (E1021K) of the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). Image result for light bulb despicable me 1000 genomes….became 100,000 genomes. Dec 2023- UK Biobank releases 500,000 whole genome database. 45  33 ? Families with the E1021K p110δ mutation. (A) Open circles and squares, unaffected; filled circles and squares, affected; partly filled circles and squares, available data indicate recurrent infections. Age at the time of death is shown for patients who died ≤30 years of age. PIK3CD genotype is shown if known. wt, wild-type allele encoding glutamic acid (E1021); mut, mutant allele encoding lysine (K1021). (B) Sequence chromatogram showing heterozygous mutation c.3061G>A in the PIK3CD gene leading to the E1021K amino acid change in p110δ. CpG dinucleotide is underlined. From-Angulo et al. Science 15 November 2013: Vol. 342 no. 6160 pp. 866-871 46  PM 34 PDGF receptor peptide= X-X-pY-X-N P p110 In vitro activity and structure of p110δ RTK p85/p110 PDGF receptor peptide +/- peptide 3X PI4,5P2 6X Same! PIP3 Active site E1021 PI3K lipid vesicle binding Mimics P.M. Lipid vesicle for assays and binding studies (includes PI4,5P2) (A) Basal and pY peptide-stimulated PI3K activity at 20-nM concentration. Graphs are mean ± SD of three independent experiments. (B) Inhibition of mutant and wild-type p110δ/p85α as a function of IC87114 or GS-1101 concentration. (C) Domain organization of p110δ. (D) Structural model of the p110δ/p85α heterodimer. p110δ catalytic subunit (pale green); nSH2 and iSH2 domains of the p85 regulatory subunit (cyan); cSH2 domain (magenta); p110δ activation loop (thick chocolate tube beneath kα12); residue E1021 of p110δ (green spheres); and the analogous residue in H1047R mutant of p110α (cyan spheres). The IC87114 inhibitor bound in the active site is shown in stick representation. (E) Membrane binding of p110δ. 47  35 Activity of the mutant PI3K ex vivo in patients’ leukocytes B and T cells + drug Take away message: Patient PIP3 levels increased in T- lymphocytes PI3K is expressed predominantly in cells of hematopoietic lineage and is the major PI3K isoform signaling downstream of T and B cell antigen receptors. They measured levels of PIP3 in both T lymphocyte lineages. They found consistently higher PIP3 levels in patients than in controls before stimulation and 10, 20, 30, and 60s. after stimulation. Immunological presentation of patients resembles this phenotype and indicates a B cell defect. An increased population of circulating transitional B cells may reflect a block in late stages of B cell maturation or an enhanced death of mature B cells. “The possibility of treating patients with p110 inhibitors should therefore be considered.” 48  Location of Phosphoinositides PtdIns is a lower abundance phospholipid found in eukaryotic cells and phosphoinositides are generally less abundant by one order of magnitude, with PtdIns(4)P and PtdIns(4,5)P2 representing the bulk of these lipids in mammalian cells. Each of the seven phosphoinositides has a unique subcellular distribution with a predominant localization in subsets of membranes, including PtdIns(4,5)P2 on the ………………………………………………………………………….……., PtdIns3P on early ………….………… PtdIns(3,5)P2 on late ……………………….… and PtdIns4P on the ……………..…………..… network (figures below). The differential intracellular distribution of phosphoinositides, together with their high turnover, makes these lipids optimal mediators of signaling. These phosphoinositides identify specific membranes (molecular markers) and allow them to recruit proteins from the cytosol that are involved in specific cellular events. PM Golgi early endosomes plasmid GFP PH domain of PLC1 binds PtdIns(4,5)P2 PtdIns(4,5)P2 PtdIns(4)P PtdIns(3)P Fluorescence micrographs illustrating the predominant localization of PtdIns(4,5)P2 (plasma membrane), PtdIns(4)P (Golgi) and PtdIns(3)P (early endosomes) in Chinese hamster ovary cells as revealed by the pleckstrin homology (PH) domain of PLC1, the PH domain of FAPP1 and the FYVE domain of Hrs (all fused to GFP, green). The nucleus is shown in blue (DAPI staining). Not shown is PtdIns(3,5)P2 which resides at late endosomes. These PH and FYVE domains selected because they are biochemically known to bind a specific PI. 49  PH domains in human proteins … there are 250 in the human genome Protein Domain architecture Specific PI ligand PI-4,5-P2 PI-4,5-P2 (GEF for Arfs) PI-3,4,5-P3 PI-3,4-P2/ /PKB PK PI-3,4,5-P3 PI-4,5-P2 PK =protein kinase 50  PH domains in human proteins Phosphorylated inositol rings of phosphoinositides form binding sites for a particular type of lipid-binding domain (PH domains), which is present on specific binding partners; PH domains have been identified in >150 different human proteins (250 domains in total). A. Protein binding to phosphoinositide 2nd messengers recruits PH domain containing proteins to cytoplasmic face of membrane, where they can interact with other membrane-bound proteins, like activators, inhibitors or substrates. = PI-3,4,5-P3 production + PDGF PKB Examples of PH domains that translocate to the PM in response to the addition of PDGF. GFP Vector with PH domain and GFP Time --> 51  Back to second messengers in Karp Some inositol-containing messengers do not remain in the membrane lipid bilayer. When acetylcholine binds to smooth muscle cell (eg. blood vessel, stomach wall), contraction occurs, the bound receptor activates a heterotrimeric G protein. The heterotrimeric G protein activates an effector, phosphatidylinositol (PI)- specific phospholipase C- (PLC). The phospholipase C activated by G proteins is identified as PLC to distinguish it from the isoform PLC (activated by receptor tyrosine kinases). PLC (at inner membrane surface) catalyzes split of PI4,5P2 into 2 molecules, both of which are second messengers important in cell signaling: inositol 1,4,5-triphosphate (………………….) & diacylglycerol (………………….). PLC is situated at inner membrane surface, bound there by interaction between its PH domain & a PI4,5P2 molecule embedded in the bilayer. Diacyglycerol – a lipid molecule that remains in membrane after its formation by PLC domain?? DAG recruits & activates an effector called protein kinase C (PKC). From Karp, Cell and Molecular Biology: 52 [10,000X] Concepts and Experiments, 4th Edition, Wiley What does inositol 1,4,5-triphosphate (IP3) do? - small, water soluble sugar phosphate; capable of rapid diffusion throughout cell interior. IP3 forms at the plasma membrane, diffuses into cytosol & binds to a specific IP3 receptor at smooth endoplasmic reticulum (SER) surface; SER is a site of …………………….……………. in a variety of cells. IP3 receptor isalso a tetrameric Ca2+ channel; IP3 binding opens the channel & allows Ca2+ ions to diffuse from SER into cytoplasm; calcium ions can also be considered as intracellular or second messengers. Ca2+ ions bind to various target molecules, triggering specific responses. Examples: smooth muscle contraction. Several Ca2+ binding protein in cells, the best known being calmodulin (CaM). Ca2+ binds via EF hands. Outside cell  specific for PLC Slide 29….and 37 [1X] From Karp, Cell and Molecular Biology: Concepts and Experiments, 4th Edition, Wiley [10,000X] 53  Note: ~10% of these protein kinases Protein Kinases are pseudokinases The human genome has approx. 518 protein kinases - 478 with typical eukaryotic protein kinase catalytic domain and 40 with atypical structure. Is it likely that these 478 evolved from a common ancestral gene as all have a common 300 aa catalytic core. 300 aa kinase domain Protein kinases comprise ~2-3% of the protein encoding portion of the genome in most eukaryotes. Human (eukaryotic) protein kinases are divided into 8 major groups: - AGC (cyclic-nucleotide (PKA, PKG) and Ca2+/phospholipids/ diacylglycerol regulated (PKC) - CaMK (Ca2+/calmodulin regulated & relatives) - CK1/2 (casein kinase) See next - CMGC (Cdks & MAPKs) pg - PTK (protein tyrosine kinases) - PTKL (tyrosine kinase-like; diverse group) - STE (MAPK cascade families) - other (not falling into other major groups) 300 aa kinase domain 258 out of 518 kinases have additional protein domains - 83 different domains characterized including: - 25 with PH domains - 23 with SH2 domains Some have additional subunits as well. ex., cyclin dependent kinases yeast (cdk1) No tyrosine kinases in yeast? (single cell Euk) Paradigm: tyrosine kinases evolved in metazoans (multicellular animals) to allow intercellular communication 35 54 T HE Human (All the protein kinases) Kinome mTOR ATM ATR DNAPK 55 F ro m www.cellsignal.com/reference/kinase/kinome.html  How do PKs recognize their substrates? Most PKs (e.g. cAMP-dependent PK, PKA) phosphorylate multiple substrate proteins, but always at well-defined sites. Sequencing of these sites revealed conserved features or motifs near the phosphorylated residue (P), especially clusters of basic or acidic residues. PKA sites usually have basic residues (Arg, Lys) at P-2, P-3. There is often, but not always, a hydrophobic residue at P+1: Peptide; X= any amino acid X-X-X-S-X-X-X P-3 P-2 ↓ P+1 arg-arg-x-ser-hyd Mg-ATP Phosphorylation consensus motif PK Peptide substrate PKA will phosphorylate small synthetic peptides (10–15 residues) containing this motif. Studies using variant peptides in which the basic residues are replaced or moved have confirmed their importance. Since such small peptides have no regular 3-D structure, conformation appears not to be important. Conformation can be important in the negative sense, since a site will not be phosphorylated unless it is exposed on the surface of the protein. Structural biology and mass spectrometry has revealed most phosphorylation sites reside on intrinsically disordered regions (IDR) of proteins. Sites phosphorylated by cAMP-dependent PK(PKA) ↓ Phosphorylase kinase  subunit: -val-glu-phe-arg-arg-leu-ser-ile- Phosphorylase kinase  subunit: -arg-thr-lys-arg-ser-asn-ser-val- Glycogen synthase site 1A: -gln-trp-pro-arg-arg-ala-ser-cys- Glycogen synthase site 1b: -gly-ser-lys-arg-ser-asn-ser-val- Slide 33 Glycogen synthase site 2: -pro-leu-ser-arg-thr-leu-ser-val- Pyruvate kinase (L type): -gly-tyr-leu-arg-arg-ala-ser-val- 6-Phosphofructo-2-kinase: -leu-gln-arg-arg-arg-gly-ser-ser- Hormone-sensitive lipase: -pro-met-arg-arg-ser-val- Protein phosphatase inhibitor-1: -ile-arg-arg-arg-arg-pro-thr-pro- RI pseudosubstrate site: -gly-arg-arg-arg-arg-gly-ala-ile- 56  Profiling the substrate specificity of the human serine/threonine kinome 303 Ser/Thr kinases Nature | Vol 613 | 26 January 2023 | 759 Profiling the substrate specificity of the human serine/threonine kinome. a, Experimental workflow for the positional scanning peptide array analysis and representative results. Z denotes fixed positions containing one of the 20 natural amino acids, or either phosphorylated Thr (pThr) or phosphorylated Tyr (pTyr). X denotes unfixed positions containing randomized mixtures of all natural amino acids except Ser, Thr and Cys. Darker spots indicate preferred residues. b, Dendrogram of the human protein kinome, highlighting the Ser/Thr kinases analysed in this study. 57  Phosphorylation motifs for the human Ser/Thr kinome enable comprehensive scoring and annotation of the human phosphoproteome. Nature | Vol 613 | 26 January 2023 | 759 Phosphorylation motifs for the human Ser/Thr kinome enable comprehensive scoring and annotation of the human phosphoproteome. The 82,735 mapped phosphorylation sites come from public databases compiling results from many studies. a, Schematic of the substrate-scoring process. Outcomes for the approach: Findings suggest that the presence of negative-selectivity elements flanking a putative phosphorylation site can be used to insulate a substrate from inappropriate phosphorylation by dozens of related kinases, whereas the absence of such negative selectivity can enable protein kinases in distinct pathways to converge on the same target. We can now use this information to link phosphorylation sites back to specific protein kinases. 2024 paper for the tyrosine kinases-…. 58  Global motif analysis reveals how kinase perturbations and pathway rewiring reshape the phosphoproteome. ↑ cAMP DNA damage secreted PK cells arrested in mitosis Nature | Vol 613 | 26 January 2023 | 759 Fig. 4 | Global motif analysis reveals how kinase perturbations and pathway rewiring reshape the phosphoproteome. a, Workflow of the motif enrichment analysis of phosphoproteomics data. All phospho-peptides are enriched and analysed by quantitative MS, and then related back to protein kinases that ‘likely’ phosphorylate these substrates. The schematic was created using BioRender. b–e, Results from published datasets. b, Conditioned medium of HepG2 cells after genetic deletion of FAM20C44. c, Cultured myotubes after 30 min treatment with 2 μM isoproterenol. d, HeLa cells after mitotic arrest by treatment for 45 min with 0.1 μM PLK1 inhibitor BI 2536. e, A549 cells 2 h after exposure to 6 Gy of ionizing radiation. 59 Protein kinases regulated by phosphorylation Some protein kinases are activated by phosphorylation outside of the kinase domain, e.g. phosphorylase kinase; the first PK to be discovered (1950’s). Consists of 4 subunits, the holoenzyme being . The  subunit is the catalytic subunit. In the absence of Ca2+, the  and/or  subunits (which are orthologous with each other) appear to inhibit . This inhibition is alleviated either by phosphorylation of  and  by cAMP-PK, or by binding of Ca2+ to , which is calmodulin: cAMP -> PKA -> PhK -> Phos b -> Phos a = glycogen degradation Purified from skeletal muscle Phosphorylated by PKA on  and Phospho-  subunits enzyme 100 Activity % max. dephospho- enzyme [Ca2+] 60  Phosphorylation within the kinase domain More commonly, phosphorylation within the kinase domain, at site(s) between the highly conserved Asp-Phe-Gly (DFG) and Ala/Ser-Pro-Glu (APE) motifs activate protein kinases. The sites (underlined) are usually threonine, but can also be serine or tyrosine. 300 aa N C DFG APE PKA DFGFAKRVKGRTWTLCGTPEYLAPE PKB/Akt DFGLCKESIHDGTVTHTFCGTIEYMAPE Regulation of MAP PKC DFGMCKEHMMDGVTTRTFCGTPDYIAPE kinases by MAP kinase phosphatases - CDK2 DFGLARAFGIPIRVYTHEVVTLWYRSPE ScienceDirect MAP kinase DFGLARIADPEHDHTGFLTEYVATRWYRAPE MAPKK(MEK) DFGVSGQLIDSMANSFVGTRSYMSPE CaMKI DFGLSKMEDPGSVLSTACGTPGYVAPE PhK DFGFSCQLDPGEKLREVCGTPSYLAPE AMPK DFGLSNMMSDGEFLRTSCGSPNYAAPE This is the activation loop, T-loop or activationsegment. -C helix N-domain Peptide substrate in active site ADP in active site Protein kinases have C-domain two domains - the N- domain and C- domain. 61  Inactive kinases commonly have misaligned -C helix, which prevents the proper positioning of ATP...Mg2+-ATP that is! Mg2+ + ATP MgATP 1 : 20 The S/T/Y-phosphate in the activation loop stimulates kinase activity by binding to basic residues in loops within the active site, which helps position catalytic residues and locking the enzyme in the active conformation. Mg2+-ATP -C Structural elements conserved in all kinases are shown as white (N-lobe) and tan/red (C-lobe) cartoons. From Taylor SS, Kornev AP. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci. 2011 36(2):65-77. 62  pT From-Taylor SS, Kornev AP. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci. 2011 36(2):65-77. P of the T-loop= repositioning of the C helix and binding of substrate MgATP….=active protein kinase 1X→ 50-100X, or even >1000-fold in some cases Basal -> Activated 63  Lipid kinases and the PIKKs That make the 7 phosphoinositides Ex. PI3K See slide 51 Catalytic domain of lipid kinases is similar to protein kinases. From Walker et al 1999 Nature p313-20. Look back at slide ~47 (case study) 64 PROTEIN PHOSPHATASES The discovery of protein phosphatases, like protein phosphorylation, originated in the study of glycogen metabolism. CLASSIFICATION OF PROTEIN PHOSPHATASES Eukaryotic protein kinases fall into a single protein family, all sharing a common catalytic core….300aa ! In contrast, protein phosphatases of eukaryotic cells come from four distinct gene families. PKA P-motif is:…………… Protein phosphatases are different from protein kinases because they do not dephosphorylate at well-defined sequences. In 1980s all of the known extra-mitochondrial protein (serine/threonine) phosphatases could be ascribed to four classes of enzymes using biochemical criteria: cation dependence and sensitivity to inhibitors. BIOCHEMICAL CLASSIFICATION OF SER/THRPROTEIN PHOSPHATASES Based on in vitro assays Protein Inhibition by Divalent Phosphatase protein: cation requirement in I1 or I2 assay? PP1 Yes None PP2A No None PP2B No Ca2+ PP2C No Mg2+ 65  NaCl gradient Ion exchange column of liver extract +Mg 2+ =PP2C +Ca2+ deP of P-S/T substrate +I1 =PP2B or I2 Column fractions Using mammalian phosphoprotein substrates, protein phosphatases with these biochemical properties have been identified in extracts of yeast, plants, algae, paramecium, vertebrates, invertebrates.......all eukaryotes. Gene family classification From cDNA and PCR cloning and genomics we can place the protein phosphatases into 4 groups based on sequence. 1. PPP………PP1, PP2A, PP2B, PP4-7 2. PPM……..PP2C 3. PTP 4. Asp-based 66  Human Protein Phosphatases 1. 2. 3. Classic deP pY only DSP= dual specificity….? deP the PIs Cell cycle (deP pY of CDKs) 4. Moorhead et al. Nature Reviews Molecular Cell Biology 8, 234– 244 (March 2007). 67 1.The PPP family (serine/threonine-specific) (a)PP1, PP2A and PP2B are related in sequence (PPP family). (b)The PPP family is highly conserved across species. -e.g. the plant versions of PP1 and PP2A catalytic subunits are almost 80% identical to the mammalian enzymes. (c) There are isoenzymes of each of PP1, PP2A and PP2B. -e.g. 3 human isoforms (genes) of PP1 and 9 Arabidopsis isoforms of PP1. (d)Novel PPP family members are known. In addition to PP1, 2A and 2B, there are other relatives in this gene family (PP4, PP5, PP6 and PP7) that are also highly conserved across species. 2. The PPM family (serine/threonine-specific) These are the Mg2+-dependent enzymes (PP2C) They are unrelated (in amino acid sequence) to the PPP family and belong to a distinct gene family. Structurally, the active sites are similar. Excellent example of convergent evolution! 68 3. The Protein Tyrosine Phosphatase (PTP) superfamily (107 genes in humans, but only 85 are active enzymes!) - all contain the motif CXXXXXR, better known as CX5R. -the catalytic signature motif C and R bind the substrate phosphate in a deep pocket that cannot be reached by phospho-serine or phospho- threonine. Note, this deep pocket is only true for the P-Tyr specific enzymes. P-Tyr P-Ser PTP active site pocket CxxxxxR CxxxxxR How were PTPs discovered? X-X-X-pY-X-X-X (peptide substrate) Salt gradient pY peptide deP A280 (protein) PTP 69  Class I PTPs o These include the CLASSIC receptor PTP and nonreceptor PTPs that only dephosphorylate tyrosine residues. Regulation of MAP kinases by MAP kinase phosphatases - ScienceDirect o Class I also includes the dual-specificity family (DSPs) MAPK cascade Mitogen Activated PK Frontiers | Mitogen-Activated Protein Kinase Cascades in Plant Hormone Signaling | Plant Science Mitogen- promotes mitosis ,ex PDGF, EGF (MEK) (MEK) First found as a factor that was activated seconds to minutes after growth factor or insulin ? addition to cells. October 2018 | https://doi.org/10.3389/fpls.2018.01387 Regulation of MAP kinases by MAP kinase phosphatases - ScienceDirect o They were given the ‘unfortunate’ name dual-specificity because the first enzyme from the family to be characterized was the MAPK-phosphatase. o This very diverse bunch [DSPs] all maintain the CX5R signature, but some dephosphorylate phosphoinositides, mRNA and even serine and threonine. PTEN, myotubularins P Y Class II PTPs o CDC25s (dephosphorylate key pY on the cyclin- dependent kinases [cdks]). CDK Class III PTPs o Low Molecular Weight PTP (LMWPTPs) 70  Transcribes DNA into mRNA, snRNA and microRNAs 4. A fourth family utilizes Asp-based catalysis FCP/SCP dephosphorylates 3 serines on RNA-polymerase II. Phosphorylation of 52 heptad repeats of YpS2TpS5pS7 of human Pol II CTD at Ser2, Ser5, and Ser7 involves multistep transcriptional regulation. The phosphorylation state of the C-terminal domain of RNA polymerase II controls transcript maturation. Transcription from RNA polymerase II (Pol II) involves docking to a pre- initiation complex and phosphorylation of the largest subunit of Pol II on its C-terminal domain (CTD). During mRNA biogenesis, specific mRNA- processing factors are recruited depending on the phosphorylation pattern of the CTD, which changes during the transcription cycle. At termination, protein phosphatases function on the CTD, allowing Pol II to be recruited to another pre- initiation complex. Moorhead et al. Nature Reviews Molecular Cell Biology 8, 234– 244 (March 2007). 71  = PP1, PP2A, PP2B, PP4-7 Inhibitors of P PP f a m i l y o f Protein Phosphatases Members of the PPP family are potently and specifically inhibited by a range of chemically-diverse, naturally occurring, tumor-promoting toxins from marine dinoflagellates, freshwater blue-green algae, soil bacteria, and insects: Okadaic acid 1982 IC50s: PP1=20 nM PP2A=0.1 nM -accumulates in filter feeding organisms Microcystin Cyclic heptapeptide Liver toxin Tumor promoter Blue green algae that cause fresh water blooms