CDK13/CDC2L5 interacts with L-type cyclins and regulates alternative splicing
ABSTRACT Due to the strong sequence homology it has been suggested that CDC2L5 and CDK12 belong to a high molecular weight subfamily of CDC2 family with PITAI/VRE motifs [F. Marques, J.L. Moreau, G. Peaucellier, J.C. Lozano, P. Schatt, A. Picard, I. Callebaut, E. Perret, A.M. Geneviere, A new subfamily of high molecular mass CDC2-related kinases with PITAI/VRE motifs, Biochem. Biophys. Res. Commun. 279 (2000) 832-837]. Recently, we reported that CDK12 interacts with L-type cyclins and is involved in alternative splicing regulation [H.-H. Chen, Y.-C. Wang, M.-J. Fann, Identification and characterization of the CDK12/Cyclin L1 complex involved in alternative splicing regulation, Mol. Cel. Biol. 26 (2006) 2736-2745]. Here, we provide evidence that CDC2L5 also interacts with L-type cyclins and thus rename it as cyclin-dependent kinase 13 (CDK13). The kinase domain of CDK13 is sufficient to bind the cyclin domains of L-type cyclins. Moreover, CDK13 and L-type cyclins modulate each other's subcellular localization. When CDK13 and an E1a minigene reporter construct were over-expressed in HEK293T cells, CDK13 alters the splicing pattern of E1a transcripts in a dose-dependent manner. Similar to effects of CDK12, effects of CDK13 on splicing pattern are counteracted by SF2/ASF and SC35. These findings strengthen CDK12 and CDK13 as a subfamily of cyclin-dependent kinases that regulate alternative splicing.
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ABSTRACT: Cdk12 and Cdk13 are Cdc2-related proteins that share 92% identity in their kinase domains. Using in situ hybridization and Western blot analysis, we detected the expression of Cdk12 and Cdk13 mRNAs and their proteins in developing mouse embryos, especially during development of the nervous system. We explored the roles of Cdk12 and Cdk13 in neuronal differentiation using the P19 neuronal differentiation model. Upon knockdown of Cdk12 or Cdk13, no effect on differentiated cell numbers was detected, but a substantial decrease of numbers of neurons with long neurites was identified. Similarly, knockdown of Cdk12 or Cdk13 in primarily cultured cortical neurons shortens the averaged axonal length. A microarray analysis was used to examine changes in gene expression after knockdown or overexpression of Cdk12 and we identified Cdk5 as a molecule potentially involved in mediating the effect of Cdk12 and Cdk13. Depletion of Cdk12 or Cdk13 in P19 cells significantly reduces Cdk5 expression at both the mRNA and protein levels. Expression of Cdk5 protein in the developing mouse brain is also reduced in conditional Cdk12-knockout mice in proportion to the residual amount of Cdk12 protein present. This suggests that the reduced axonal outgrowth after knockdown of Cdk12 or Cdk13 might be due to lower Cdk5 expression. Furthermore, overexpression of Cdk5 protein in P19 cells was able to partially rescue the neurite outgrowth defect observed when Cdk12 or Cdk13 is depleted. Together, these findings suggest that Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression.Experimental Neurology 07/2014; 261. DOI:10.1016/j.expneurol.2014.06.024 · 4.62 Impact Factor
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ABSTRACT: Cyclin-dependent kinases (CDKs) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that play critical roles in the control of cell-cycle progression, transcription, and neuronal functions. However, the functions, substrates, and regulation of many CDKs are poorly understood. To systematically investigate these features of CDKs, we conducted a proteomic analysis of the CDK family and identified their associated protein complexes in two different cell lines using a modified SAINT (Significance Analysis of INTeractome) method. The mass spectrometry data were deposited to ProteomeXchange with identifier PXD000593 and DOI 10.6019/PXD000593. We identified 753 high-confidence candidate interaction proteins (HCIPs) in HEK293T cells and 352 HCIPs in MCF10A cells. We subsequently focused on a neuron-specific CDK, CDK5, and uncovered two novel CDK5-binding partners, KIAA0528 and fibroblast growth factor (acidic) intracellular binding protein (FIBP), in non-neuronal cells. We showed that these three proteins form a stable complex, with KIAA0528 and FIBP being required for the assembly and stability of the complex. Furthermore, CDK5-, KIAA0528-, or FIBP-depleted breast cancer cells displayed impaired proliferation and decreased migration, suggesting that this complex is required for cell growth and migration in non-neural cells. Our study uncovers new aspects of CDK functions, which provide direction for further investigation of these critical protein kinases.Molecular & Cellular Proteomics 08/2014; 13(11). DOI:10.1074/mcp.M113.036699 · 7.25 Impact Factor
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ABSTRACT: Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported.03/2015; 7(1):179-237. DOI:10.3390/cancers7010179