Binding Specificity for RACK1 Resides in the V5 Region of βII Protein Kinase C

Stebbins, Elizabeth G.; Mochly‐Rosen, Daria

doi:10.1074/jbc.m101044200

Cited by 170 publications

(164 citation statements)

References 46 publications

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“…The V1 domain of ⑀PKC is homologous to the C2 domain of the ␤PKC (1). However, there is an additional RACK-binding site in the V5 region of ␤PKC (38) and molecular dynamics studies with the C2 region of ␤PKC showed that an intramolecular interaction between the RACK and the RACK-binding site in the C2 region is not possible (39). Instead we suggest that the intramolecular interaction between the RACK and the RACK-binding site in ␤PKC is likely to occur between the C2 and V5 regions in ␤PKC.…”

Section: Mathematical Modeling Of ⑀Pkc Translocation Suggests That ⑀Pmentioning

confidence: 82%

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Schechtman

Craske

Kheifets

et al. 2004

Journal of Biological Chemistry

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Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive ⑀PKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed⑀RACK. An amino acid difference between the ⑀RACK sequence in ⑀PKC and its homologous sequence in ⑀RACK constitutes a change from a polar non-charged amino acid (asparagine) in ⑀RACK to a polar charged amino acid (aspartate) in ⑀PKC. Here we show that mutating the aspartate to asparagine in ⑀PKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone-or PMAinduced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to ⑀RACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the ⑀RACK site and RACK-binding site within ⑀PKC is critical and rate limiting in the process of PKC translocation.The protein kinase C (PKC) 1 family of phospholipid (PL) -dependent serine/threonine kinases undergoes a conformational change and translocation, or movement, from the cytosolic to the cell particulate fraction upon activation (1, 2). Conformational changes in PKC from an inactive to an active state results in exposure of domains required for PKC anchoring to the particulate fraction and in increased sensitivity of the enzyme to proteases (1-3, 41). Therefore, the inactive state exists in a closed conformation, with the proteolytic sites protected, whereas the active state is in an open conformation with exposed proteolytic sites. Structural alterations from the closed to open states involve disruption of intramolecular interactions within the enzyme.An intramolecular interaction in inactive PKC between the catalytic site and a site in the regulatory domain that resembles a substrate phosphorylation site but lacks a serine or threonine phosphoacceptor (pseudosubstrate site) has been previously identified (3, 6). Deletion of the pseudosubstrate ( -substrate) site generated a constitutively active enzyme (6) and mutations of the basic residues in the -substrate site reduced the affinity of the catalytic site to the -substrate site generating a constitutively active enzyme, preferentially localized to the cell particulate fraction (6). Furthermore, conversion of the alanine in the -substrate site to a glutamic acid, mimicking a phosphorylated amino acid, resulted in loss of binding of the -substrate site to the catalytic site, creating a constitutively active enzyme (6). Finally, a peptide corresponding to the -substrate site is a competitive inhibitor of PKC catalytic activity (6).We previously demonstrated that translocation of PKC is associated with binding of each activated PKC isozyme to...

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Section: Mathematical Modeling Of ⑀Pkc Translocation Suggests That ⑀Pmentioning

confidence: 82%

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Schechtman

Craske

Kheifets

et al. 2004

Journal of Biological Chemistry

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“…Additionally, PKCβII was found at different subcellular locations in cardiac myocytes [91], Stebbins and Mochly-Rosen found that it binds specifically to RACK1 in vitro through the V5 region [92]. It is also known to impact signaling pathways in a specific manner as shown with its specific activation of the MAPK pathway [93].…”

Section: Pkcβiimentioning

confidence: 99%

Specific protein kinase C isoforms as transducers and modulators of insulin signaling

Sampson

Cooper

2006

Molecular Genetics and Metabolism

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Recent studies implicate specific PKC isoforms in the insulin-signaling cascade. Insulin activates PKCsα, βII, δ and ζ in several cell types. In addition, as will be documented in this review, certain members of the PKC family may also be activated and act upstream of PI3 and MAP kinases. Each of these isoforms has been shown one way or another either to mimic or to modify insulin-stimulated effects in one or all of the insulin-responsive tissues. Moreover, each of the isoforms has been shown to be activated by insulin stimulation or conditions important for effective insulin stimulation. Studies attempting to demonstrate a definitive role for any of the isoforms have been performed on different cells, ranging from appropriate model systems for skeletal muscle, liver and fat, such as primary cultures, and cell lines and even in vivo studies, including transgenic mice with selective deletion of specific PKC isoforms. In addition, studies have been done on certain expression systems such as CHO or HEK293 cells, which are far removed from the tissues themselves and serve mainly as vessels for potential protein-protein interactions. Thus, a clear picture for many of the isoforms remains elusive in spite of over two decades of intensive research. The recent intrusion of transgenic and precise molecular biology technologies into the research armamentarium has opened a wide range of additional possibilities for direct involvement of individual isoforms in the insulin signaling cascade. As we hope to discuss within the context of this review, whereas many of the long soughtafter answers to specific questions are not yet clear, major advances have been made in our understanding of precise roles for individual PKC isoforms in mediation of insulin effects. In this review, in which we shall focus our attention on isoforms in the conventional and novel categories, a clear case will be made to show that these isoforms are not only expressed but are importantly involved in regulation of insulin metabolic effects.

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“…Myristoylated PKC peptides from the carboxyl terminus of the V5 region of PKC ␣ (myr-PQFVHPILQSAV-amide), PKC ␤I (myr-DQNE-FAGFSYTNPEFVINV-amide), or PKC ␤II (myr-SFVNSEFLKPEVKSamide) were added to a final concentration of 100 M 30 min before the addition of apoptotic thymocytes. The myristate moieties coupled to the amino terminus of these peptides allow membrane permeability, permitting their use in primary cells (34). All inhibitors were nontoxic at the times and concentrations utilized, as determined by the lactate dehydrogenase cytotoxicity detection kit.…”

Section: Methodsmentioning

confidence: 99%

Activation of Protein Kinase C βII by the Stereo-specific Phosphatidylserine Receptor Is Required for Phagocytosis of Apoptotic Thymocytes by Resident Murine Tissue Macrophages

Todt¹,

Hu²,

Punturieri³

et al. 2002

Journal of Biological Chemistry

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We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AMø) and peritoneal macrophages (PMø) and that such phagocytosis is markedly lower in AMø compared with PMø. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two Mø populations. By immunoblotting, AMø expressed equivalent PKC but lower amounts of other isoforms (␣, ␤I, ␤II, ␦, ⑀, , and ), with the greatest difference in ␤II expression. A requirement for PKC ␤II for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC ␤II, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC ␤II myristoylated peptide as a blocker. Exposure of PMø to phosphatidylserine (PS) liposomes specifically induced translocation of PKC ␤II and other isoforms to membranes and cytoskeleton. Both AMø and PMø expressed functional PS receptor, blockade of which inhibited PKC ␤II translocation. Our results indicate that murine tissue Mø require PKC ␤II for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in Mø phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC ␤II activation.

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Binding Specificity for RACK1 Resides in the V5 Region of βII Protein Kinase C

Cited by 170 publications

References 46 publications

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

Specific protein kinase C isoforms as transducers and modulators of insulin signaling

Activation of Protein Kinase C βII by the Stereo-specific Phosphatidylserine Receptor Is Required for Phagocytosis of Apoptotic Thymocytes by Resident Murine Tissue Macrophages

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