Activation of PKC through GPCR
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Activation of PKC through GPCR
PKC (Protein Kinase-C) is a cyclic nucleotide-independent enzyme that phosphorylates serine and threonine residues in many target proteins. PKC plays a pivotal role in mediating cellular responses to extracellular stimuli involved in proliferation, differentiation, apoptosis, and exocytotic release in a number of non-neuronal systems such as Islet cells, Chromaffin cells and Paramecium. PKC has also been implicated in phosphorylation of several neuronal proteins, which are thought to regulate neurotransmitter release and establish long-term potentiation in memory formation. PKC is not a single enzyme but a family of serine/threonine kinases. At least eleven closely related PKC isozymes have been reported that differ in their structure, biochemical properties, tissue distribution, subcellular localization, and substrate specificity. The PKC family has been divided into three groups: cPKC (Conventional PKC) isoforms (comprising Alpha, Beta and Gamma), that require Calcium and DAG (Diacylglycerol) for activation; nPKC (Novel PKC) isoforms (comprising Delta, Epsilon, Eta{also known as PKC-L}, Theta and Mu {the mouse homolog of human PKC-Mu is known as PKD}) that require DAG; and aPKC (Atypical PKC) isoforms, namely Zeta and Iota, that require neither Calcium nor DAG. A new PKC member has recently been discovered and is referred to as PKC-Nu. It contains 890 amino acid residues and exhibits highest sequence similarity to PKC-Mu/PDK thereby posing the possibility of a fourth subfamily of PKCs, comprising these isoforms (Ref.1 & 2).

All PKCs possess a Phospholipid-binding domain for membrane interaction. The general structure of a PKC molecule consists of a C (Catalytic) and an R (Regulatory) domain composed of a number of conserved regions, interspersed with regions of lower homology, the variable domains. Generally, PKC isozymes contain four conserved regions termed C1-C4. C1 contains a Cysteine-rich motif and forms the DAG binding site. The autoinhibitory pseudosubstrate sequence is upstream of the cysteine-rich motif in the same region. C2 contains the recognition site for acidic lipids and, in some isozymes, the Ca2+ binding site. C3 and C4 form the ATP and substrate binding lobes of the kinase, respectively. In its unstimulated state, most of the PKC resides in the cytosol. In this state, the pseudosubstrate sequence of the regulatory domain of PKC interacts with the Catalytic domain and prevents access of the substrate to the catalytic site. Inactive PKC is not freely distributed throughout the cytoplasm but appears localized to specific sites within the cell. This localization is facilitated by association of PKC with scaffolding proteins, such as AKAP79 (A Kinase-Anchoring Protein-79) and Gravin. AKAP79 binds a number of PKC isoforms including Alpha, BetaII, Delta, Epsilon, and Zeta (Ref. 3).

PKC activation occurs when Plasma membrane receptors Like GPCRs (G-Protein Coupled Receptors) coupled to PLC are activated, releasing DAG. The binding of hormones, growth factors, neurotransmitters, and other agonists of GPCR results in activation of PLC or PLA2 (Phospholipase-A2) via a G-Protein-dependent phenomenon. Heterotrimeric G-Proteins consist of Alpha, Beta and Gamma subunits that are stably associated in the inactive, GDP-bound state. Physical interaction between a G-Protein and an agonist-occupied receptor triggers the exchange of GDP for GTP on the Alpha subunit and the subsequent dissociation of this subunit from the tightly associated Beta Gamma dimer. Both the G-Alpha-GTP and G-Beta Gamma entities participate in PLC activation and PKC signaling. Different PLC isozymes take part in the activation of PKC. PLC-Beta isozymes are activated by the G-AlphaQ or G-BetaGamma subunit released from heterotrimeric G-Protein 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. 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, activated by G-AlphaS, appears to be responsible for the phosphorylation of PLC-Gamma1 in vascular smooth muscle cells and platelets. GPCR also activates PLC-Delta isoforms. 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-Delta1activity. The second route involves a protein similar to Rho-GAP that was also shown to stimulate PLC-Delta1 activity. The recently identified human 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. G-AlphaS activates Rap via AC (Adenylate Cyclase), cAMP (Cyclic Adenosine 3,5-monophosphate) and EPAC (Exchange Protein Activated by cAMP). Rap further activates PLC-Epsilon. 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 G-Alpha13 (Ref. 4, 5 & 6).

Phosphoinositide-specific PLC1 family of enzymes are responsible for the hydrolysis of PIP2 (Phosphatidylinositol 4,5-bisphosphate) that results in the generation of second messengers InsP3 or IP3 (Inositol 1,4,5- trisphosphate) and DAG. The IP3 causes the release of endogenous Ca2+ that binds to the cytosolic PKC and exposes the phospholipid binding site. The binding of Ca2+ translocates PKC to the membrane, where the C1 and C2 domains interact with DAG and Phosphatidylserine, respectively. Phosphatidylserine is the membrane lipid anchor for both cPKCs and nPKCs, although other membrane phospholipids may ultimately link extracellular signals to intracellular events through PKC. This interaction causes the pseudosubstrate domain to dissociate from the catalytic domain, which results in activation of PKC. Specific anchoring proteins (immobilized at particular intracellular sites) localize the Kinase to its site of action. These proteins include RACKS (Receptors for Activated C-Kinase) and Adducins. PKC can also be activated by PI3K (Phosphoinositide-3 Kinase). The hormone Relaxin binds to its GPCR receptors LGR7/LGR8 (Leucine-Rich Repeat Containing G-Protein Coupled Receptor-7/8) and activates G-Beta Gamma subunit of G-Proteins, leading to the activation of PI3K. PI3K enhance cAMP production by activating atypical PKC: PKC-Zeta. PKC-Zeta activates Type II and V AC (Adenylate Cyclase), which further activates cAMP. cAMP modulates cardiac contractility and release of metabolic energy by converting to AMP (Adenosine Monophosphate) in presence of PDE (Phosphodiesterase) (Ref.6 & 7).

Activated PKC phosphorylates various substrates in the pathway, triggering various cellular responses, including, secretion, changes in membrane permeability, and gene 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 (Myosin Light Chain Phosphatase), causing inhibition of MLCP activity, which further leads to MLC 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. 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. Altered PKC activity has been linked with various types of malignancies. Higher levels of PKC and differential activation of various PKC isozymes have been reported in breast tumors, adenomatous pituitaries, thyroid cancer tissue, leukemic cells, and lung cancer cells. Downregulation of PKC-Alpha is reported in the majority of colon adenocarcinomas and in the early stages of intestinal carcinogenesis. Thus, PKC inhibitors have become important tools in the treatment of cancers. The involvement of PKC in the regulation of apoptosis adds another dimension to the effort to develop drugs that will specifically target PKC (Ref.8, 9 & 10).