The substrates and binding partners of protein kinase Cε

Wang

et al. 2016

Cell Physiol Biochem

Background/Aims: Phorbol myristate acetate (PMA) exerts a pleiotropic effect on the growth and differentiation of various cells. Protein kinase Cs (PKCs) plays a central role in mediating the effects of PMA on cells. The present study investigated whether the down-regulation of protein kinase C-ε (PKC-ε) is involved in the inhibition of vascular smooth muscle cell (VSMC) proliferation caused by prolonged PMA incubation. Methods: Using cell counting, Cell Counting Kit-8 (CCK-8) and EdU incorporation assay on VSMCs, we evaluated the inhibitory effects of prolonged incubation of PMA, of lentiviruses carrying the short-hairpin RNAs (shRNA) of PKC-ε and of the PKC-ε inhibitor peptide on the proliferation and viability of cells. The effect of PKC-ε down-regulation on growth of rat breast cancer SHZ-88 cells was also measured. Results: The prolonged incubation of VSMCs with PMA for up to 72 hours resulted in attenuated cell growth rates in a time-dependent manner. The expression of PKC-ε, as assessed by Western blotting, was also decreased accordingly. Notably, the number of EdU-positive cells and the cell viability of VSMCs were decreased by shRNA of PKC-ε and the PKC-ε inhibitor peptide, respectively. The proliferation of rat breast cancer SHZ-88 cells was also attenuated by lentivirus-induced shRNA silencing of PKC-ε. Conclusions: Prolonged incubation of PMA can inhibit the expression of PKC-ε. The effect results in the inhibition of VSMC proliferation. PKC-ε silencing can also attenuate breast cancer cell growth, suggesting that PKC-ε may be a potential target for anti-cancer drugs.

Section: Discussionsupporting

confidence: 81%

Down-Regulation of Protein Kinase C-ε by Prolonged Incubation with PMA Inhibits the Proliferation of Vascular Smooth Muscle Cells

Wang

et al. 2016

Cell Physiol Biochem

Journal of Biological Chemistry

“…When stimulated by PMA, isoforms of PKC regulate the machinery that controls the intracellular trafficking and transport of proteins; for example, PKC⑀ stimulates membrane trafficking through binding of ␤'COP, a coatomer protein, and regulates the function of cytoskeletal components (25). We and others have shown that PMA stimulates the trafficking of MT1-MMP to the plasma membrane (17,26).…”

Section: Pma Stimulates Mt1-mmp Phosphorylation-phorbolmentioning

confidence: 99%

Phosphorylation of Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) and Its Vesicle-associated Membrane Protein 7 (VAMP7)-dependent Trafficking Facilitate Cell Invasion and Migration

Williams¹,

Coppolino

2011

108

132

Background: Intracellular trafficking of MT1-MMP is essential for its role in tumor cell invasion. Results: Mutation of Thr 567 in MT1-MMP altered its internalization and recycling and associated biochemical signaling. Conclusion: Phosphorylation of MT1-MMP at Thr 567 regulates its intracellular trafficking, which is coupled to integrin trafficking and ERK phosphorylation. Significance: Phosphorylation of MT1-MMP may be a regulatory point for control of tumor cell invasion.

“…20 Specifically, PKCe, unlike other PKC family members, contains an F-actinbinding region that promotes the formation of F-actin filaments by preventing depolymerization and increasing the rate of actin filament elongation. 21 During mitotic cytokinesis of several cell types (ie, COS-7, HeLa, MEF, HEK 293), PKCe assembles with 14-3-3zeta, and together they transiently accumulate at the actomyosin ring with RhoA and is required to dissociate both itself and RhoA from the midbody. 22 In neurons, PKCe promotes neurite outgrowth, which can be blocked by forced activation of the ROCK/RhoA pathway, indicating that PKCepilson may attenuate RhoA/ROCK pathway activity in the process.…”

Section: Introductionmentioning

confidence: 99%

Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition

Gobbi

Mirandola

Carubbi

et al. 2013

Blood

• PKCe, regulating RhoA activity, is a critical mediator of proplatelet formation. • PKCe shut down results inRhoA expression levels that are incompatible with normal platelet generation.During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kinase Cepsilon (PKC«), a member of the protein kinase C family of serine/threonine-kinases, expression varies during human megakaryocyte differentiation and modulates megakaryocyte maturation and platelet release. Here we used an in vitro model of murine platelet production to investigate a potential role for PKC« in proplatelet formation. By immunofluorescence we observed that PKC« colocalizes with a/b-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKC« expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKC« inhibition resulted in lower proplatelet numbers and larger diameter platelets in culture as well as persistent RhoA activation. Finally, we demonstrate that pharmacological inhibition of RhoA is capable of reversing the proplatelet defects mediated by PKC« inhibition. Collectively, these data indicate that by regulating RhoA activity, PKC« is a critical mediator of mouse proplatelet formation in vitro. (Blood. 2013;122(7):1305-1311) IntroductionDefective platelet numbers can compromise proper wound healing and cause bleeding, whereas excessive platelet production or activation can lead to thrombosis. As a result, platelet homeostasis is a tightly regulated process. The process of generating thousands of platelets from a single megakaryocyte is characterized by several morphologically distinct stages that involve extensive cytoskeletal remodeling. In particular, megakaryoctyes, following polyploidization and prior to platelet release, form elongated cellular processes called proplatelets (proPLT).1 Perturbations in proPLT formation lead to alterations in platelet morphology and thrombocytopenia, 2,3 emphasizing the importance of this step. The marginal microtubule band, actin-based cytoskeleton, and spectrin-based membrane skeleton are all prominent components of the cytoskeletal remodeling that occurs during platelet formation. 4 Specifically, dynamic tubulin polymerization is required for the proper platelet morphology, assembly of the marginal microtubule coil, proPLT extensions and microtubule band formation.4-8 Various mutations in genes that regulate actin polymerization and turnover cause thrombocytopenia due to altered formation and morphology of proPLT exten...