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Protein kinase Czeta regulates Cdk5/p25 signaling during myogenesis.

ABSTRACT: Atypical protein kinase Czeta (PKCzeta) is emerging as a mediator of differentiation. Here, we describe a novel role for PKCzeta in myogenic differentiation, demonstrating that PKCzeta activity is indispensable for differentiation of both C2C12 and mouse primary myoblasts. PKCzeta was found to be associated with and to regulate the Cdk5/p35 signaling complex, an essential factor for both neuronal and myogenic differentiation. Inhibition of PKCzeta activity prevented both myotube formation and simultaneous reorganization of the nestin intermediate filament cytoskeleton, which is known to be regulated by Cdk5 during myogenesis. p35, the Cdk5 activator, was shown to be a specific phosphorylation target of PKCzeta. PKCzeta-mediated phosphorylation of Ser-33 on p35 promoted calpain-mediated cleavage of p35 to its more active and stable fragment, p25. Strikingly, both calpain activation and the calpain-mediated cleavage of p35 were shown to be PKCzeta-dependent in differentiating myoblasts. Overall, our results identify PKCzeta as a controller of myogenic differentiation by its regulation of the phosphorylation-dependent and calpain-mediated p35 cleavage, which is crucial for the amplification of the Cdk5 activity that is required during differentiation.

SUBMITTER: de Thonel A

PROVIDER: S-EPMC2854099 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CDK5 activator protein p25 preferentially binds and activates GSK3?.

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| S-EPMC4234543 | biostudies-literature

The adapter protein ZIP binds Grb14 and regulates its inhibitory action on insulin signaling by recruiting protein kinase Czeta.

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Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity.

Project description:Aberrant cell-cycle activity and DNA damage are emerging as important pathological components in various neurodegenerative conditions. However, their underlying mechanisms are poorly understood. Here, we show that deregulation of histone deacetylase 1 (HDAC1) activity by p25/Cdk5 induces aberrant cell-cycle activity and double-strand DNA breaks leading to neurotoxicity. In a transgenic model for neurodegeneration, p25/Cdk5 activity elicited cell-cycle activity and double-strand DNA breaks that preceded neuronal death. Inhibition of HDAC1 activity by p25/Cdk5 was identified as an underlying mechanism for these events, and HDAC1 gain of function provided potent protection against DNA damage and neurotoxicity in cultured neurons and an in vivo model for ischemia. Our findings outline a pathological signaling pathway illustrating the importance of maintaining HDAC1 activity in the adult neuron. This pathway constitutes a molecular link between aberrant cell-cycle activity and DNA damage and is a potential target for therapeutics against diseases and conditions involving neuronal death.

| S-EPMC2912147 | biostudies-literature

Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

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| S-EPMC4288260 | biostudies-literature

p32 is a novel mammalian Lgl binding protein that enhances the activity of protein kinase Czeta and regulates cell polarity.

Project description:Lgl (lethal giant larvae) plays an important role in cell polarity. Atypical protein kinase C (aPKC) binds to and phosphorylates Lgl, and the phosphorylation negatively regulates Lgl activity. In this study, we identify p32 as a novel Lgl binding protein that directly binds to a domain on mammalian Lgl2 (mLgl2), which contains the aPKC phosphorylation site. p32 also binds to PKCzeta, and the three proteins form a transient ternary complex. When p32 is bound, PKCzeta is stimulated to phosphorylate mLgl2 more efficiently. p32 overexpression in Madin-Darby canine kidney cells cultured in a 3D matrix induces an expansion of the actin-enriched apical membrane domain and disrupts cell polarity. Addition of PKCzeta inhibitor blocks apical actin accumulation, which is rescued by p32 overexpression. p32 knockdown by short hairpin RNA also induces cell polarity defects. Collectively, our data indicate that p32 is a novel regulator of cell polarity that forms a complex with mLgl2 and aPKC and enhances aPKC activity.

| S-EPMC2064465 | biostudies-literature

Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Czeta (PKCzeta) and regulates PKCzeta activity.

Project description:Regulation of cell polarity is an important biological event that governs diverse cell functions such as localization of embryonic determinants and establishment of tissue and organ architecture. The Rho family GTPases and the polarity complex Par6/Par3/atypical protein kinase C (PKC) play a key role in the signaling pathway, but the molecules that regulate upstream signaling are still not known. Here we identified the guanine nucleotide exchange factor ECT2 as an activator of the polarity complex. ECT2 interacted with Par6 as well as Par3 and PKCzeta. Coexpression of Par6 and ECT2 efficiently activated Cdc42 in vivo. Overexpression of ECT2 also stimulated the PKCzeta activity, whereas dominant-negative ECT2 inhibited the increase in PKCzeta activity stimulated by Par6. ECT2 localization was detected at sites of cell-cell contact as well as in the nucleus of MDCK cells. The expression and localization of ECT2 were regulated by calcium, which is a critical regulator of cell-cell adhesion. Together, these results suggest that ECT2 regulates the polarity complex Par6/Par3/PKCzeta and possibly plays a role in epithelial cell polarity.

| S-EPMC444862 | biostudies-literature

The carboxyl-terminal domain of atypical protein kinase Czeta binds to ceramide and regulates junction formation in epithelial cells.

Project description:Atypical protein kinase Cs (PKCs) (aPKCzeta and lambda/iota) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCzeta and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCzeta (C20zeta, amino acids 405-592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131-180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20zeta protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20zeta-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20zeta) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20zeta binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.

| S-EPMC2682895 | biostudies-literature

No difference in kinetics of tau or histone phosphorylation by CDK5/p25 versus CDK5/p35 in vitro.

Project description:CDK5/p35 is a cyclin-dependent kinase essential for normal neuron function. Proteolysis of the p35 subunit in vivo results in CDK5/p25 that causes neurotoxicity associated with a number of neurodegenerative diseases. Whereas the mechanism by which conversion of p35 to p25 leads to toxicity is unknown, there is common belief that CDK5/p25 is catalytically hyperactive compared to CDK5/p35. Here, we have compared the steady-state kinetic parameters of CDK5/p35 and CDK5/p25 towards both histone H1, the best known substrate for both enzymes, and the microtubule-associated protein, tau, a physiological substrate whose in vivo phosphorylation is relevant to Alzheimer's disease. We show that the kinetics of both enzymes are the same towards either substrate in vitro. Furthermore, both enzymes display virtually identical kinetics towards individual phosphorylation sites in tau monitored by NMR. We conclude that conversion of p35 to p25 does not alter the catalytic efficiency of the CDK5 catalytic subunit by using histone H1 or tau as substrates, and that neurotoxicity associated with CDK5/p25 is unlikely attributable to CDK5 hyperactivation, as measured in vitro.

| S-EPMC2840295 | biostudies-literature

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