Author: Michelle Lum

Publisher:

Published: 2012

Total Pages: 188

ISBN-13:

Protein Kinase C (PKC) is a family of AGC kinases (a family including the A, G, and C protein kinases) that play critical roles in regulation of cell growth and cell cycle progression, differentiation, cell survival and apoptosis, gene expression, receptor trafficking and desensitization, and cell transformation. As regulators of fundamental cellular processes, the activity of members of this family is tightly controlled. While mechanisms of activation of these proteins have been extensively characterized, the pathways underlying signal termination are less well understood. PKCs are divided into 3 classes based on structure and coactivator requirements. Classical PKC (cPKC: PKC alpha, PKC beta I, PKC beta II and PKC gamma) are activated by diacylglycerol (DAG), Ca2+ and phosphatidylserine, while novel PKC (nPKC) do not require Ca2+ and atypical PKC (aPKC) are independent of both DAG and Ca2+.^The difference in cofactor requirements for these isozymes is a result of differences in their C1 and C2 regulatory domain structures. The C1 domain is involved in DAG binding, while the C2 domain is involved in Ca2+ binding. Ligand binding by a number of growth factor and cytokine receptors leads to activation of phospholipase C and subsequent generation of DAG and inositide trisphosphate (which mobilizes intracellular Ca2+). The accumulation of DAG leads to recruitment of cPKCs and nPKCs to the plasma membrane where they are activated due to displacement of a pseudosubstrate domain from the active site. In addition to activation by physiological signals, cPKCs and nPKCs can also be activated by pharmacological agonists that bind to the same site as DAG. These agonists include phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), and bryostatins, such as bryostatin-1 (bryo). Newly synthesized PKCs are not competent for activation but require priming phosphorylation on three conserved sites on the activation loop, turn motif and hydrophobic motif. These priming phosphorylations, which occur in an ordered manner, render the kinase competent for activation and stabilize it against denaturation and degradation. Acute termination of signaling is achieved by the rapid metabolism of DAG (by e. g., diacylglycerol kinase). This loss of signal leads to "reverse translocation" of membrane bound PKC to the cytoplasm in a process that is dependent on kinase activity. Pharmacological agonists are not subject to rapid metabolism and thus elicit a sustained activation of PKCs. In addition to acute termination of signal, it has long been recognized that prolonged activation of PKCs (either by pharmacological agonists or sustained physiological stimuli) leads to desensitization of this signaling system via degradation of the protein. Although activation-induced loss of PKC expression has been observed in many systems, there is considerable confusion regarding the mechanism(s) underlying agonist-induced desensitization of PKC signaling. Part of this confusion may stem from the extensive use of overexpression systems to analyze aspects of PKC regulation. While these systems can yield valuable information, our analysis indicates that overexpression can drive activated PKC isozymes into alternate pathways of degradation, which do not reflect those utilized by the endogenous proteins. Studies detailed herein analyze various mechanisms of desensitization of PKC signaling that are relevant to the endogenous protein by examining the effects of various agonists on the localization and processing of cellular PKC alpha.^The first part of this work used a combination of biochemical and immunolocalization studies to examine the mechanisms underlying a novel non-proteasomal pathway of degradation that is induced by prolonged bryo treatment in various cell types. Bryo initially induces translocation of endogenous PKC alpha; to the plasma membrane, where a pool of the protein is subjected to proteasomal degradation. A second subpopulation is internalized through a clathrin-independent, but cholesterol- and genistein-sensitive pathway, which involves trafficking through EEA1-positive early endosomes and Rab7-positive late endosomes/multivesicular bodies. The ultimate fate of internalized PKC alpha is degradation by lysosomal proteases in the perinuclear region. Analysis of the effect of endolysosomal disrupting agents in multiple cell lines points to lysosomal processing of activated PKC alpha as a common mechanism for its desensitization. The second part of this thesis explored the role of dephosphorylation and heat shock proteins (Hsps) in agonist-induced proteasomal degradation of endogenous PKC alpha. A widely accepted mechanism for stimulus-induced downregulation of PKCs involves priming site dephosphorylation, which targets the protein for proteasomal degradation. However, studies described here demonstrate that PKC agonists induce downregulation of endogenous PKCalpha with minimal accumulation of non-phosphorylated enzyme in multiple cell types. Furthermore, all or most of the non-phosphorylated enzyme detected in agonist-treated cells results from delayed maturation rather than dephosphorylation of the protein. Thus, PKC agonists induce at most low levels of dephosphorylation of endogenous PKC alpha;, and dephosphorylation is not a prerequisite for enzyme degradation. Analysis of the functions of Hsp90 and Hsp70/Hsc70 revealed distinct roles for these chaperones in regulating agonist-induced PKC alpha; degradation. Hsp90 prevents dephosphorylation of the activated enzyme and protects the mature, phosphorylated form of protein from proteasomal clearance following activation. In contrast, while Hsp70/Hsc70 also protects PKC alpha; from dephosphorylation, it enhances degradation of activated PKC alpha; by facilitating proteasomal processing of mature phosphorylated protein. Notably, downregulation of non-phosphorylated enzyme showed little dependence on Hsp70/Hsc70, suggesting that mature and non-phosphorylated species are targeted for proteasomal degradation via different pathways. Finally, lysosomal degradation of PKC alpha is not dependent on Hsps or the phosphorylation state of the enzyme. The third part of this work examines desensitization of PKC signaling following stimulation by DAG, the major physiological activator of cPKC and nPKC isozymes.^Analysis of the effects of a single addition of various DAGs (which are rapidly metabolized), and of short term pulse treatment with phorbol esters, confirmed that acute reversal of PKC alpha signaling following loss of signal involves dissociation of fully phosphorylated PKC alpha from the plasma membrane and cytosolic accumulation of the enzyme, via a mechanism that is dependent on PKC alpha activity. In contrast, repeated addition of DAGs resulted in sustained association of PKC alpha with the plasma membrane, in a pattern comparable with that induced by the phorbol ester PMA. Unlike the effects of PMA, however, chronic DAG stimulation failed to promote degradation/downregulation of PKC alpha, although downregulation of PKC delta and epsilon was readily apparent. Priming site dephosphorylation of PKC alpha was also not observed and Hsp70/Hsc70 and Hsp90 were excluded as regulators of PKC alpha phosphorylation and stability in this context. Importantly, while long-term activation of PKC alpha by DAG did not lead to degradation of the enzyme, it did induce desensitization of PKC alpha signaling, as seen by reversal of PKC alpha mediated effects on ERK activation, p21Waf1/Cip1 induction, and Id1 and cyclin D1 downregulation. These findings point to a previously undefined mechanisms for desensitization of PKC alpha, which can be attributed either to direct effects on PKC alpha function or to alterations in its downstream signaling pathways. Through analysis of the endogenous protein, each of these studies has identified a novel mechanism for desensitization of PKC alpha signaling. Collectively, the data confirm that the phosphorylated species of PKC alpha is a direct target for proteasomal and lysosomal degradation, and highlight the existence of multiple mechanisms for processing activated PKCs in cells.