PKC isozymes

PKC is a family of enzymes whose members play central roles in transducing information from external stimuli to cellular responses. Members of this family of serine/ threonine kinases respond to signals that cause lipid hydrolysis. PKC isozymes phosphorylate an abundance of substrates, leading to both short-term cellular responses such as regulation of membrane transport and long-term responses such as memory and learning. 

Signaling by PKC is terminated by concentrations of its ligands dropping to basal levels (i.e., Ca2+ and diacylglycerol) and by dephosphorylation of the three processing sites. Dephosphorylation is controlled, in part, by a recently discovered hydrophobic phosphorylation motif phosphatase. This phosphatase, PHLPP (for PH domain Leucine-rich repeat Protein Phosphatase) dephosphorylates conventional and novel PKC isozymes, initiating their downregulation. 

PKC isozymes are involved in a wide array of diverse cellular functions [5]. Most isozymes (e.g., PKC (3) are involved in proliferative responses, and hyperactivation with phorbol esters most typically results in cell growth and differentiation. However, isozymes can have opposing functions. PKC 8 is well-characterized as an apoptotic kinase, whereas the closely related PKC e is antiapoptotic. PKC also plays a key role in learning and memory. 

Conventional and novel PKC isozymes are potently activated by phorbol esters, heterocyclic compounds found in the milky sap exuded by plants of the Euphorbiaccae family. This sap was used medicinally as a counterirritant and cathartic agent over the millennia we now know that the active ingredients, phorbol esters, specifically bind to the Cl domain, the diacylglycerol sensor described above. In fact, their ability to recruit PKC to membranes is so effective that phorbol esters cause maximal activation of conventional PKCs, bypassing the requirement for Ca2+. This module is found in a number of other proteins in addition to PKC, so the profound effects of phorbol esters on cells are mediated by other proteins in addition to PKC. 

Upon activation, neurons begin trafficking TRPVl to the membrane where the receptors become activated, desensitized and then recycled to the intracellular compartments. Translocation of TRPVl to the cell membrane occurs via SNARE (snapin and synaptotagmin IX)-mediated exocytosis [37]. Broadly speaking, activation involves phosphorylation by protein kinases (most notably, protein kinase A [PKA] and C [PKC]) and desensitization involves de-phosphorylation by phosphatases (e.g. calcineurin) [38]. Among PKC isozymes, PKCp seems to be of particular importance [39]. 

As described in several monographs [4], bryostatin 1 exhibits significant in vitro and in vivo antineoplastic activity against a range of tumor cell lines including murine leukemia, B-cell lymphoma, reticulum cell sarcoma, ovarian carcinoma, and melanoma. It is also effective in the modulation of apoptotic function [5], the reversal of multidrug resistance [6], and stimulation of the immune system [7]. These unique features displayed by bryostatin 1 are attributed to its high affinity for protein kinase C (PKC) isozymes and its ability to selectively modulate their functions [8]. PKCs are a type of intracellular serine and threonine kinase that... 

FIGURE 20-8 Linear representation of PKC isozymes. See text for details. Reproduced with permission from Tan, S. L. and Parker, R J. Biochemical Journal 376 545-552,2003 [23] The Biochemical Society. 

From overexpression studies, it can be inferred that individual isoforms of PKC are precisely directed to distinct subcellular locations (e.g. PKCa to the endoplasmic reticulum and PKCS to the Golgi). Directing PKC isozymes to specific subcellular loci appears to occur via interaction of the enzyme with localized intracellular binding proteins. Such proteins may or may not be substrates for PKC. An example of the latter category would be RACK (receptors for activated C kinase) 1. RACKs are thought to interact only with activated PKCs and to direct translocated PKCs to specific loci. 

To date, there have only been a limited number of studies directly examining PKC in bipolar disorders [77], Although undoubtedly an oversimplification, particulate (membrane) PKC is sometimes viewed as the more active form of PKC, and thus an examination of the subcellular partitioning of this enzyme can be used as an index of the degree of activation. Friedman etal. [78] investigated PKC activity and PKC translocation in response to serotonin in platelets obtained from bipolar-disorder patients before and during lithium treatment. They reported that the ratios of platelet-membrane-bound to cytosolic PKC activities were elevated in the manic patients. In addition, serotonin-elicited platelet PKC translocation was found to be enhanced in those patients. With respect to brain tissue, Wang and Friedman [74] measured PKC isozyme levels, activity and translocation in postmortem brain tissue from patients with bipolar disorder, and reported increased PKC activity and translocation in the brains of bipolar patients compared with controls, effects which were accompanied by elevated levels of selected PKC isozymes in cortices of bipolar disorder patients. 

Evidence accumulating from various laboratories has clearly demonstrated that lithium, at therapeutically relevant concentrations, exerts significant effects on the PKC signaling cascade. Current data suggest that chronic lithium attenuates PKC activity, and down-regulates the expression of PKC isozymes V in the frontal cortex and hippocampus [79, 80], Chronic lithium has also been demonstrated to dramatically reduce the hippocampal levels of a major PKC substrate, myristoylated-alanine-rich C kinase substrate (MARCKS), which has been implicated in regulating long-term neuroplastic events. 

Although these effects of lithium on PKC isozymes and MARCKS are striking, a major problem inherent in neu-ropharmacologic research is the difficulty in attributing therapeutic relevance to any observed biochemical finding. It is thus noteworthy that the structurally dissimilar... 

Geng WD, Boskovic G, Fultz ME, Li C, Niles RM, Ohno S, Wright GI. Regulation of expression and activity of four PKC isozymes in confluent and mechanically stimulated UMR-108 osteoblastic cells. J Cell Physiol. 2001 189 216-228. 

The protein kinase C (PKC) signaling pathway has been associated with modulation of A-methyl-D-aspartate (NMDA) receptor activity, motor behavior, learning, and memory, all of which are severely impaired in intoxication with sarin and similar OPs. There was a reduction in the immunoreactivity levels of betall-PKC and Zeta-PKC in the frontal cortex (up to 24 h), and in the striatum (up to 5 days) post-sarin exposure, in contrast to the increase in the immunoreactivity of both enzymes in the hippocampus or thalamus, following a IxLDso exposure to sarin. These observations suggest a role for both conventional and atypical PKC isozymes in OP-induced neuropathy in the rat and further support their role in cell death (Bloch-Shilderman et al., 2005). 

Inhibition of the PKC isozyme superfamily elicits apoptosis in tumor cells. Phenethyl isothiocyanate (PEITC) conjugates of sphingosine and sphinganine (Fig. 13) exert potent antineoplas-tic effects in human leukemia HL-60 cells by the inhibition of conventional PKC/novel PKC activity and ERK1/ERK2 activity... 

PAF receptor is coupled to the activation of both protein kinase C and tyrosine kinase (Shukla, 1992, Izumi and Shimizu, 1995). PAF receptor antagonists block both pathways. The activation of PKC is through the Imown PLC generated diglyceride -t- Ca pathway (leyasu et. al. 1982). Activation of PKC in platelets (eg. by phrobol myristate acetates) inhibited PAF stimulation of platelets. On the other hand PKC inhibitors (eg. staurosporine) enhanced PAF stimulated IP, production. In rabbit platelets PKC activation by PAF was suggested to be independent of enzyme translocation (Pelech et. al. 1990). Human platelets have 6 PKC isozymes (alpha, p, 8, zeta, eta and theta). PAF stimulated 200% and 175% increase in the levels of membrane - bound PKC eta and theta... 

The best known receptors for phorbol esters and their derivatives are the isozymes of protein kinase C (PKC), which bind phorbol esters and the physiological second messenger diacylglycerol (DAG) by cysteine-rich domains, the Cl domains. The exact functions of the different PKC isozymes is not known at present however, they have been shown to be involved in synaptic transmissions, the activation of ion fluxes, secretion, cell cycle control, differentiation, proliferation, tumorigenesis, metastasis, and apoptosis. 

Both PLDl and PLD2 can be activated by PKC in intact cells [62, 63], whereas only PLDl can be activated in vitro [11, 12, 47]. However, this difference may reflect the in vitro assay conditions rather than an intrinsic difference between the two isozymes. The ability of phorbol esters such as PMA to activate PLD in vivo has been demonstrated in many cell types [62-64]. The role of PKC isozymes in this effect has been shown by the inhibitory effects of relatively selective PKC inhibitors such as Ro-31-8220, bisindolylmaleimide I, G66076, calphostin C, bryosta-tin and cheleryfhrine [53]. Most of these inhibitors target fhe ATP binding site of... 

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