Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ACCESS DATA

Json Xml

Similar Datasets

CDK5 activator protein p25 preferentially binds and activates GSK3?.

Project description:Glycogen synthase kinase 3? (GSK3?) and cyclin-dependent kinase 5 (CDK5) are tau kinases and have been proposed to contribute to the pathogenesis of Alzheimer's disease. The 3D structures of these kinases are remarkably similar, which led us to hypothesize that both might be capable of binding cyclin proteins--the activating cofactors of all CDKs. CDK5 is normally activated by the cyclin-like proteins p35 and p39. By contrast, we show that GSK3? does not bind to p35 but unexpectedly binds to p25, the calpain cleavage product of p35. Indeed, overexpressed GSK3? outcompetes CDK5 for p25, whereas CDK5 is the preferred p35 partner. FRET analysis reveals nanometer apposition of GSK3?:p25 in cell soma as well as in synaptic regions. Interaction with p25 also alters GSK3? substrate specificity. The GSK3?:p25 interaction leads to enhanced phosphorylation of tau, but decreased phosphorylation of ?-catenin. A partial explanation for this situation comes from in silico modeling, which predicts that the docking site for p25 on GSK3? is the AXIN-binding domain; because of this, p25 inhibits the formation of the GSK3?/AXIN/APC destruction complex, thus preventing GSK3? from binding to and phosphorylating ?-catenin. Coexpression of GSK3? and p25 in cultured neurons results in a neurodegeneration phenotype that exceeds that observed with CDK5 and p25. When p25 is transfected alone, the resulting neuronal damage is blocked more effectively with a specific siRNA against Gsk3? than with one against Cdk5. We propose that the effects of p25, although normally attributed to activate CDK5, may be mediated in part by elevated GSK3? activity.

| S-EPMC4234543 | biostudies-literature

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

Project description:Grb14 is a member of the Grb7 family of adapters and acts as a negative regulator of insulin-mediated signaling. Here we found that the protein kinase Czeta (PKCzeta) interacting protein, ZIP, interacted with Grb14. Coimmunoprecipitation experiments demonstrated that ZIP bound to both Grb14 and PKCzeta, thereby acting as a link in the assembly of a PKCzeta-ZIP-Grb14 heterotrimeric complex. Mapping studies indicated that ZIP interacted through its ZZ zinc finger domain with the phosphorylated insulin receptor interacting region (PIR) of Grb14. PKCzeta phosphorylated Grb14 under in vitro conditions and in CHO-IR cells as demonstrated by in vivo labeling experiments. Furthermore, Grb14 phosphorylation was increased under insulin stimulation, suggesting that the PKCzeta-ZIP-Grb14 complex is involved in insulin signaling. The PIR of Grb14, which also interacts with the catalytic domain of the insulin receptor (IR) and inhibits its activity, was preferentially phosphorylated by PKCzeta. Interestingly, the phosphorylation of Grb14 by PKCzeta increased its inhibitory effect on IR tyrosine kinase activity in vitro. The role of ZIP and Grb14 in insulin signaling was further investigated in vivo in Xenopus laevis oocytes. In this model, ZIP potentiated the inhibitory action of Grb14 on insulin-induced oocyte maturation. Importantly, this effect required the recruitment of PKCzeta and the phosphorylation of Grb14, providing in vivo evidences for a regulation of Grb14-inhibitory action by ZIP and PKCzeta. Together, these results suggest that Grb14, ZIP, and PKCzeta participate in a new feedback pathway of insulin signaling.

| S-EPMC139806 | biostudies-literature

Structure-activity relationship study of 2,4-diaminothiazoles as Cdk5/p25 kinase inhibitors.

Project description:Cdk5/p25 has emerged as a principle therapeutic target for numerous acute and chronic neurodegenerative diseases, including Alzheimer's disease. A structure-activity relationship study of 2,4-diaminothiazole inhibitors revealed that increased Cdk5/p25 inhibitory activity could be accomplished by incorporating pyridines on the 2-amino group and addition of substituents to the 2- or 3-position of the phenyl ketone moiety. Interpretation of the SAR results for many of the analogs was aided through in silico docking with Cdk5/p25 and calculating protein hydrations sites using WaterMap. Finally, improved in vitro mouse microsomal stability was also achieved.

| S-EPMC3062192 | biostudies-literature

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.

Project description:Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3? and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research.

| S-EPMC4288260 | 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

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

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

Cyclin-dependent kinase 5 (CDK5) regulates the circadian clock.

Project description:Circadian oscillations emerge from transcriptional and post-translational feedback loops. An important step in generating rhythmicity is the translocation of clock components into the nucleus, which is regulated in many cases by kinases. In mammals, the kinase promoting the nuclear import of the key clock component Period 2 (PER2) is unknown. Here, we show that the cyclin-dependent kinase 5 (CDK5) regulates the mammalian circadian clock involving phosphorylation of PER2. Knock-down of Cdk5 in the suprachiasmatic nuclei (SCN), the main coordinator site of the mammalian circadian system, shortened the free-running period in mice. CDK5 phosphorylated PER2 at serine residue 394 (S394) in a diurnal fashion. This phosphorylation facilitated interaction with Cryptochrome 1 (CRY1) and nuclear entry of the PER2-CRY1 complex. Taken together, we found that CDK5 drives nuclear entry of PER2, which is critical for establishing an adequate circadian period of the molecular circadian cycle. Of note is that CDK5 may not exclusively phosphorylate PER2, but in addition may regulate other proteins that are involved in the clock mechanism. Taken together, it appears that CDK5 is critically involved in the regulation of the circadian clock and may represent a link to various diseases affected by a derailed circadian clock.

| S-EPMC6890458 | biostudies-literature

Regulation of cyclic AMP-dependent protein kinase levels during skeletal myogenesis.

Project description:We showed previously that the levels of type I regulatory subunit of cyclic AMP-dependent protein kinase increase during differentiation of L6 skeletal myoblasts as a result of a specific decrease in its rate of degradation. Studies on the rates of degradation of the catalytic subunit show that unlike the type I regulatory subunit, catalytic subunit is degraded very slowly in myoblasts (t1/2 = 29 h) and more rapidly in myotubes (t1/2 = 14 h). As with the regulatory subunit, the degradation of catalytic subunit is increased by treatment of myoblasts with cyclic AMP analogues. These results suggest that the overall increase in the amount of type I cyclic AMP-dependent protein kinase holoenzyme during myogenesis is due to the increase in levels of mRNA for the catalytic subunit. This probably leads to an increase in the amount of catalytic subunit, which then stabilizes the regulatory subunit, thereby causing an increase in the levels of this protein also.

| S-EPMC1133579 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data