BioC325 Lecture 5 2023 PDF

Effector Systems and Second Messengers Downstream of GPCRs (Part2) PLC-(IP3 & DAG)-PKC pathway 1  AC-cAMP-PKA pathway  PLC-(IP3 & DAG)-PKC pathway 2 Voet and Voet, 4th edition, chapter 19, p726 Fig19-5...

Effector Systems and Second Messengers Downstream of GPCRs (Part2) PLC-(IP3 & DAG)-PKC pathway 1  AC-cAMP-PKA pathway  PLC-(IP3 & DAG)-PKC pathway 2 Voet and Voet, 4th edition, chapter 19, p726 Fig19-54. Role of PIP2 in Intracellular Signaling 3  One Hydrophobic Substrate Hydrophobic  tethers to cell membrane water-soluble  cytosol 4  Four families based on sequence similarities: 1. PLCβ : most important 2. PLCγ 3. PLCδ : prototype 4. PLCε 5  Considered as prototype  Is the simplest PLC isoform  Made of: ◦ PH domain ◦ EF-hand motif ◦ C2 domain ◦ X-Y catalytic domains 6  isoforms No EF Could hand motif bind Ras 7  allows its binding to Gαq and its tethering to membranes 8  Could be activated by Gq/11, Gi, and Go ◦ Gq/11 signaling: activated by binding to α- subunit (Gαq/11) (ex: Bradykinin receptor) ◦ Gi and Go signaling: activated by binding to βγ-subunits (ex: acetylcholine M2 receptor) 9  Smooth muscle contraction Vesicle release 10 https://classes.lt.unt.edu/Fall_2009/CECS_5030_020/lsd0034/project/pages/page2.html 1. Initial attachment of PLCβ to cell membrane through its PH domain (binds PI(4,5)P2) 2. The C2 domain reinforces attachment to the membrane in Ca2+-dependent manner 3. exposing the catalytic Substrate: site (Y) to its substrate PI(4,5)P2 4. PLCβ interacts additionally with Gβγ or Gαq to initiate catalysis 11  Downstream of RTKs (ex: EGFR)  Specific for this isoform ◦ One SH3 domain ◦ Two SH2 domains  Interacts with phosphotyrosine of RTKs through its SH2 domains gets activated 12  Diacylglycerol (DAG)  Ca2+  Phorbol 12-myristate 13-acetate (PMA) 13 http://courses.washington.edu/conj/gprotein/ip3.htm 14  Phorbol esters: tumor inducers [PMA; 12-O-tetradecanoyl phorbol-13-acetate (TPA)]  First studies: croton oil (skin-irritant and tumor-promoting) extracted from indian plant  Mechanism: They compete with DAG on its binding site activate PKC 15  PKC: cyclic nucleotide-independent protein kinase  First isolated from bovine cerebellum in 1970s 16  Human PKC family consists of 15 isozymes classified into 3 subfamilies based on their structural domains and requirements for activation  Conventional: cPKC ❖c for conventional ❖4 isoforms  Novel: nPKC ❖n for novel ❖3 isoforms  Atypical: aPKC ❖a for atypical ❖4 isoforms 17 1. Two Regulatory Domains (vary between PKC subfamilies): ◦ C2 domain: binds Ca2+and a phosphoinositide (phosphatidylserine: PS) ◦ C1 domain (C1a and C1b): zinc finger domain that binds DAG or PMA 2. Catalytic Domain (C3 and C4) (common for all PKC subfamilies): ◦ Ser/Thr kinase domain with a:  characteristic ATP-binding N-lobe  a segment on its C-lobe that must Regulatory be phosphorylated on a Thr to Domains enable catalysis (C1 and C2) 3. A pseudo-substrate domain C-lobe Catalytic Domains 4. A variable domain (C3 and C4) 18  Family of lipid-regulated Ser/Thr kinases  Regulated by: Ca2+ and/or lipids  PKC isozymes translocate to the plasma membrane 19 20  C= conserved CR = cystein rich domain V = variable PS = pseudosubstrate C1 domain (cystein rich) contains zinc finger domain that forms the binding site for DAG, phosphatidyl serine, and phorbol esters (ex: PMA) Each of C1a and C1b binds 2 ions of Zn2+ 21  aPKC: 1.No C2 domain No calcium binding 2. Also lacks a part of C1 domain insensitive to DAG and phorbol ester 3. only requirement for activation for aPKC: binding phosphatidyl Serine (PS) 22  Sequence of PKC Activation Auto-(P)~ 1 2 Auto-(P)~ 3 Active C2 domain 6 5 4 23 PS: phosphatidyl serine  PKC as an inactive precursor is probably associated to the cytoskeleton Accessible to ATP 1: phosphorylation by PKC kinase (also named phosphoinositide- dependent kinase 1: PDK1) at Thr500 2: autophosphorylation at Thr641 3: autophosphorylation at Ser660 1+2+3:  active C2 domain 4: C2 binds Ca2+ helps PKC translocate to membrane 5: PKC is now tethered to phosphatidyl serine on the cell membrane (also binding calcium) through C2. PKC binds to DAG through C1 6: pseudosubstrate domain at the N-terminal of PKC is released and the catalytic region C4 is ready to phosphorylate its substrate at Ser/Thr residue  PKC does not get activated until the pseudosubstrate domain is released from its N-terminal 24 C1 C3 C4 C2 PS 25 It is now thought that binding of PKC to DAG (and Ca2+) improves its membrane association and increases the chances of its recognition and phosphorylation by PDK1 and thus the subsequent activation of its catalytic domain. 26 Some PKCs could not translocate to plasma membrane by themselves  need the help of anchoring proteins. These could be PDZ- or non-PDZ-containing proteins. 27 Anchoring proteins position individual PKCs into the precise locations in which they can respond to specific receptor-mediated activating signals and address their substrates 28  InaD  Example: binding of eye-PKC to InaD in the rhodopsin receptor signaling cascade 29  RICKS/AKAPs: ◦ Receptors for inactivated C-kinases ◦ Role: differentially localize different PKC isoforms to areas of the cell  STICKs: ◦ Substrates that interact with inactive C- kinase ◦ Play role in linking actin cytoskeleton to plasma membrane  RACKs: ◦ Receptors for activated C-kinases ◦ Role:act as scaffolds by complexing multiple proteins and bringing specific PKC isoforms into close proximity with substrates  PICKs: ◦ Proteins interacting with atypical C-kinase example InaD ◦ Role: they direct PKC to its upstream activators 30  PKC is activated by: ◦ Binding of DAG, Ca2+, or phorbol esters phosphorylation and autophosphorylation  PKC is down-regulated by: ◦ Prolonged stimulation with phorbol esters: dephosphorylation and degradation of PKC ◦ DAG kinases: phosphorylate DAG to give phosphatidic acid:  DAG stores are depleted and this terminates membrane localization of PKC 31 1. Calcium Signaling 2. Proteins associated with GPCR: a. RGS (Regulators of G-Protein signaling) b. GRK (G-Receptor Kinases)