Phospholipase-C Pathway
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Phospholipase-C Pathway
Inositol lipid-specific PLC (Phospholipase-C) isozymes are key signaling proteins in the cellular action of many hormones, neurotransmitters, growth factors, and other extracellular stimuli. PLC are soluble proteins that are partly cytosolic and partly associated with membrane. The PLC family in human is comprised of 13 subtypes. On the basis of their structure, they have been divided into six classes, PLC-Beta (Beta1, 2, 3 and 4), PLC-Gamma (Gamma 1 and 2), PLC-Delta (Delta 1, 3 and 4), PLC-Epsilon, PLC-Zeta and PLC-Eta (Eta1 and 2) types. The molecular weights of each are 85kDa for the Delta form, 120-155kDa for both the Beta and Gamma forms, and 230-260kDa for the Epsilon form. These groups also differ in the mechanisms by which the isozymes are activated in response to ligand interaction with various receptors. Positive regulation mechanisms of PLC by association with membrane receptors are well characterized in Beta- and Gamma-type isozymes (Ref.1).

PLC-Beta isozymes are activated by the GN-AlphaQ or G-BetaGamma subunit released from heterotrimeric G-proteins after ligand stimulation. PLC-Beta2 and PLC-Beta3 are also activated by GTP-bound Rac (Ras-Related C3 Botulinum Toxin Substrate) isozymes. Upstream, heterotrimeric G-proteins are activated by GPCRs (G-Protein Coupled Receptors); the equivalent guanine nucleotide exchange factors for Rho-family GTPases such as Rac1 (Ras-Related C3 Botulinum Toxin Substrate-1) are RhoGEFs. Certain RhoGEFs, such as the P-Rex isozymes are also responsive to G-BetaGamma, allowing for direct crosstalk between the two classes of GTPases prior to their activation of PLC-Beta isozymes. This crosstalk most likely has biological consequences for controlling events downstream of PLC-Beta activation. PLC-Gamma isozymes, on the other hand, are activated by the phosphorylation of specific tyrosine residues through the activation of receptor or non-receptor tyrosine kinases. Polypeptide growth factors, such as PDGF (Platelet-Derived Growth Factor), EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), NGF (Nerve Growth Factor), VEGF (Vascular Endothelial Growth Factor), Glial cell-derived growth factor, and HGF (Hepatocyte Growth Factor) activates PLC-Gamma1 in a wide variety of cells. The binding of a growth factor to its receptor results in dimerization of receptor subunits, stimulation of the intrinsic Tyrosine Kinase activity of the receptor, and autophosphorylation of the receptor on specific tyrosine residues. These phosphorylated residues initiate cellular signaling by acting as high-affinity binding sites for the SH2 domains of various effector proteins. Phosphorylation of PLC-Gamma1 by PDGF, EGF, FGF, and NGF receptors occurs at identical sites: tyrosine residues 771, 783, and 1254. Growth factor induced activation of PLC-Gamma1 requires not only PLC-Gamma1 tyrosine phosphorylation but also the association of the enzyme with the Growth Factor Receptor. PLC-Gamma can also be activated by BCR (B-Cell Receptor), TCR (T-Cell Receptor) and the high-affinity IgE receptor (Fc-EpsilonRI), and the IgG receptors (Fc-GammaRs) in Hematopoietic cells. Hematopoietic cells express a variety of antigen and Ig (Immunoglobulin) receptors that are able to bind specifically to a wide spectrum of ligands. Such binding results in a rapid activation of signaling events such as the tyrosine phosphorylation and subsequent activation of PLC-Gamma1 or PLC-Gamma2 (Ref. 2, 3 & 4).

Ligation of the TCR triggers the activation of Lck (Lymphocyte-Specific Protein-Tyrosine Kinase) and Fyn (Fyn Oncogene Related to Src, FGR, YES) by unknown mechanisms. Either or both of these Src family PTKs (Protein Tyrosine Kinases) then phosphorylates tyrosine residues within ITAM sequences located in TCR and CD3 chains. Two phosphorylated tyrosine residues with this motif serve as binding sites for the tandem SH2 domains of ZAP70 (Zeta-Chain-Associated Protein Kinase). Lck or Fyn then phosphorylates the bound ZAP70, resulting in its activation. Together with Lck and Fyn, activated ZAP70 phosphorylates various downstream substrates, including membrane-bound LAT (Linker for Activation of T-Cells) and SLP76 (SH2 Domain-Containing Leukocyte Protein-76). The interaction of the N-SH2 domain of PLC-Gamma1 with a phosphorylated tyrosine residue of LAT serves to position the unphosphorylated enzyme close to activated ZAP70 and Lck or Fyn, resulting in the phosphorylation and activation of PLC-Gamma1 and in its localization in the vicinity of its substrate. Phosphorylated LAT also associates with GADS, which might in turn associate with ITK (IL-2 inducible T-cell Kinase)-bound SLP76; the close proximity of ITK and PLC-Gamma1 may result in the phosphorylation of PLC-Gamma1 by ITK (Ref. 5).

BCR engagement triggers the activation of Lyn by an unknown mechanism. Activated Lyn phosphorylates tyrosine residues within ITAM sequences located in the Ig-Alpha and Ig-Beta chains. The two phosphorylated tyrosines within this motif serve as binding sites for the tandem SH2 domains of SYK (Spleen Tyrosine Kinase), and the ITAM-bound SYK is phosphorylated (activated) by Lyn. Activated SYK then phosphorylates the cytosolic protein BLNK (B-Cell Linker Protein), thereby inducing its translocation to the cell membrane. The membrane-bound, phosphorylated BLNK also contributes to recruitment of BTK (Bruton Agammaglobulinemia Tyrosine Kinase) and PLC-Gamma2 through the binding of its phosphorylated tyrosine residues to the SH2 domains of these enzymes. The recruited BTK is phosphorylated (activated) by SYK, and activated BTK phosphorylates PLC-Gamma2. SYK also might directly phosphorylate PLC-Gamma2. The products of PI3K (PtdIns 3-Kinase) also contribute to PLC-Gamma activation in response to ligation of Fc receptors, a family of receptors that bind to soluble Ig and immune complexes via the Fc region of Ig. Both SLP76 and BLNK may also play important roles in Fc-GammaR-mediated PLC-Gamma activation. Although GPCRs lack intrinsic PTK activity, tyrosine phosphorylation of PLC-Gamma also occurs in response to ligation of several such receptors, including those for Acetylcholine (muscarinic), Angiotensin II, Thrombin, Platelet-activating factor, and ATP. c-Src appears to be responsible for the phosphorylation of PLC-Gamma1 in vascular smooth muscle cells and platelets. Alternative mechanisms for the activation of PLC-Gamma that do not rely on tyrosine phosphorylation appear to exist. PA (Phosphatidic Acid) has been shown to activate both tyrosine-phosphorylated and unphosphorylated forms of PLC-Gamma1 to similar extents by increasing their affinity for substrate vesicles. Phosphatidic Acid is an immediate product of PC (Phosphatidylcholine) hydrolysis by PLD (Phospholipase-D), activation of PLD in cells may also result in activation of PLC-Gamma. Unsaturated fatty acids, such as AA (Arachidonic Acid), also stimulate PLC-Gamma activity independently of tyrosine phosphorylation in the presence of various splicing variants of the microtubule-associated protein Tau. Although Tau is expressed exclusively in neurons, non-neuronal cells also contain a protein that, together with AA, activates PLC-Gamma. This activating protein was recently identified as a 680-kDa molecule termed AHNAK (giant in Hebrew). Tau interacts with all three of the activation reaction components: PLC-Gamma, AA, and PIP2 (Phosphatidylinositol-4,5-Bisphosphate). Bradykinin-mediated PLC-Gamma isozyme activation results in activation of PKC. cPLA2 (Phospholipase-A2) is sequentially stimulated by activation of PKC, leading the release of AA. A concerted action of central repeated units of AHNAK and AA induces PLC-Gamma1 activation PLC-Gamma is also activated by Integrins via c-Src (Ref.6, 7 & 8).

The mechanism by which PLC-Delta isoforms are coupled to membrane receptors is poorly understood. However, previous work suggests regulation of PLC-Delta1 by two distinct mechanisms. The first mechanism requires the high molecular weight GTP-binding protein Gh (also known as Transglutaminase II), which was shown to stimulate PLC-Delta1 activity. The second route involves a protein similar to RhoGAP that was also shown to stimulate PLC-Delta1 activity. Nevertheless, the mechanism of PLC-Delta1 activation is poorly understood but is believed to involve increases in intracellular Calcium concentrations. Ral (RalA and RalB), a member of the Ras superfamily of small GTPases, binds and promotes PLC-Delta1 activity in vitro and in vivo. Ral is accepted as a downstream target of Ras-p21 through activation of RalGDS, while the role of PLC-Delta1 is believed to be that of amplifying or sustaining IP3 production initiated by upstream PLC-Beta, Gamma, and - Epsilon isoforms. Ral also bind CaM in a Ca2+-dependent and -independent manner. CaM binds and inhibits PLC-Delta1 activity and Ral can reverse this inhibition. The recently identified human PLC-Epsilon has been detected in two alternatively spliced forms with molecular sizes of 260 kDa (2303 residues) and 230 kDa (1998 residues). PLC-Epsilon might be a GDP-GTP exchange factor for, as well as an effector of Ras. More specifically, PLC-Epsilon possesses a RasGEF domain that activates Ras and Rap (Ras-Related Protein) isozymes; these activated GTPases directly stimulate the Phospholipase activity of PLC-Epsilon through direct binding to the RA domains in a classical feed-forward loop. Ras is also activated downstream of receptor tyrosine kinases, whereas Rap isozymes are activated downstream of GPCRs. The phospholipase activity of PLC-Epsilon is also enhanced through the direct interaction with GTP-RhoA. Certain RhoGEFs that activate RhoA, such as p115RhoGEF, are responsive to G-Alpha12 and 13. PLC-Zeta, a sperm protein that shows similarity to a truncated PLC-Delta with the PH domain deleted, is involved in the triggering of Ca++ oscillations in eggs; however, it remains to be documented whether PLC-Zeta does have PLC enzymatic activity. The new PLC-Eta family seems to be neuron-specific and its expression profile suggests an important role postnatally in the brain. One unique feature of members of PLC-Eta family is their Ca2+ sensitivity, which is greater than that observed for PLC-Delta1. In neurons, members of the PLC-Eta family could function as Ca2+-sensor enzymes that are activated by small increases in intracellular Ca2+ concentrations under physiological conditions (Ref.8, 9, 10 & 11).

The PLC class of enzymes is involved in the signaling pathway in which a cellular response such as proliferation or secretion is produced consequent to an extracellular stimulus. Activation of mammalian phosphoinositide-specific PLC results in the hydrolysis of PIP2 to release the second messengers DAG (1,2-Diacylglycerol) and IP3 (Inositol Trisphosphate). DAG is the physiological activator of PKC (Protein Kinase-C), and IP3 diffuses through the cytosol and releases stored Ca2+ (Calcium) ions from the ER (Endoplasmic Reticulum). IP3 triggers Ca2+ mobilization. Ca2+ release activates Calm (Calmodulin) which further activates Caln (Calcineurin), CamKKs and CamKs (CamK4 and CamK2). Caln facilitates NFAT (Nuclear Factor of Activated T-Cells) translocation to the nucleus, a process that is quite essential for axonal growth. CamK4 and CamK2 phosphorylate CBP (CREB Binding Protein) and Histone Deacetylases, HDAC4, HDAC5 and HDAC7, which mediates some nuclear Ca2+ signals. HDAC export allows MEF2 (Myocyte Enhancing Factor-2) to activate transcription by recruiting other Ca2+-sensitive transcriptional factors such as NFAT (Nuclear Factor of Activated T-Cells) and transcriptional coactivators such as p300. CREB (cAMP Response Element-Binding Protein) can be phosphorylated by CamK4 at a number of sites other than Ser133, including Ser129, Ser142 and Ser143. Phosphorylation of Ser142 and dephosphorylation of Ser133 residue by CamK represses CREB activity (Ref.12 & 13).

DAG activates PKC, which in turn lead to phosphorylation of various substrate proteins that are involved in an array of cellular events. DAG may also be involved in the activation of LFA1 (Lymphocyte Function-associated Antigen-1) and thus may promote cell-cell interaction during T cell activation. PKC phosphorylates an endogenous 17 kDa inhibitory protein, CPI17 (PKC-activated 17 kDa inhibitor protein of type 1 Phosphatase). Phosphorylation of CPI17 greatly augments its ability to bind to the catalytic subunit of MLCP, causing inhibition of MLCP activity, which further leads to MLC (Myosin Regulatory Light Chain) phosphorylation that takes part in Actomyosin assembly contraction. PKC phosphorylates various transcription factors, such as NF-KappaB (Nuclear Factor-KappaB), regulating the transcription of certain genes (Elk1) and thus controlling cell proliferation or apoptosis. PKC also phosphorylates MARCKS (Myristoylated Alanine Rich-C Kinase Substrate) in response to Integrin signaling, which is involved in the reorganization of the actin cytoskeleton. PKCs also activate the ERK (Extracellular Signal-Regulated Kinase) cascade, including direct phosphorylation of either MEK (MAPK/ERK Kinase) or Raf1 (v-Raf1 Murine Leukemia Viral Oncogene Homolog-1). Although the phosphorylation of MEK by PKCs is controversial, Raf1 is phosphorylated by PKCs at multiple sites. For example, PKC phosphorylates Raf1 at serine 499. Rkip inhibits MAPK (Mitogen-Activated Protein Kinase) activation in response to growth factors or PKC activators in neuronal cells and PKC can also regulate Raf1 signaling through phosphorylation of Rkip. Classical and atypical PKCs phosphorylate Rkip on a serine residue, Ser-153, which results in the displacement of Rkip from Raf1. In addition to hydrolyzing PIP2, PI-PLC can also utilize PI (Phosphatidylinositol) or PI-4-Phosphate as substrates. Substantial progress has been achieved over the last several years with regard to the knowledge of the mechanisms by which signals are conveyed from receptors at the plasma membrane to the various PLC isozymes. This progress is largely attributable to the understanding of the modular function of regulatory domains that recognize either membrane-associated proteins or lipid molecules. One overriding principle that has emerged is that receptor-induced recruitment of PLC enzymes to the vicinity of the cell membrane achieved through such regulatory domains appears key for regulation of PLC (Ref.14 & 15).

 

References

  1. G-protein-coupled receptor agonists activate endogenous phospholipase Cepsilon and phospholipase Cbeta3 in a temporally distinct manner
  2. Regulation of phospholipase C isozymes by ras superfamily GTPases
  3. Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production
  4. IP3 receptor and calcium signaling
  5. Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current
  6. PLCbeta2-independent behavioral avoidance of prototypical bitter-tasting ligands
  7. Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction
  8. Phospholipase C-gamma: diverse roles in receptor-mediated calcium signaling
  9. Structural basis for recognition of the T cell adaptor protein SLP-76 by the SH3 domain of phospholipase Cgamma1
  10. Regulation of Phospholipase C-gamma2 Networks in B Lymphocytes
  11. Targeting of protein kinase C-epsilon during Fcgamma receptor-dependent phagocytosis requires the epsilonC1B domain and phospholipase C-gamma1
  12. AHNAK-mediated activation of phospholipase C-gamma1 through protein kinase C
  13. Role of phospholipase C-zeta domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations
  14. Role of the PKC/CPI-17 pathway in enhanced contractile responses of mesenteric arteries from diabetic rats to alpha-adrenoceptor stimulation
  15. Role of the protein kinase C-epsilon-Raf-1-MEK-1/2-p44/42 MAPK signaling cascade in the activation of signal transducers and activators of transcription 1 and 3 and induction of cyclooxygenase-2 after ischemic preconditioning