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Demonstration of cyclin-dependent kinase inhibitory serine/threonine kinase in bovine thymus
1Medical Research Council Group in Signal Transduction, University of Calgary, Calgary, Alberta, T2N 4N1 Canada.
Abstract:
A synthetic peptide corresponding in sequence to residues 6-20 of p34cdc2, cdc2(6-20), and a substitution analogue, cdc2(6-20)F15K19 , which contains Thr-14 as the only phosphorylation target were used as substrates to identify a novel protein kinase in bovine thymus cytosol. The kinase catalyzed the phosphorylation of Thr-14 in both peptides and was purified extensively on the basis of its peptide phosphorylation activity. Upon SDS-polyacrylamide gel electrophoresis analyses, the purified samples consistently displayed a prominent 43-kDa protein band which could undergo in gel autophosphorylation, thus suggesting that this band represented the kinase protein. The suggestion was supported further by the observation that both cdc2(6-20) peptide phosphorylation and the autophosphorylation reaction of the 43-kDa protein were inhibited by millimolar concentrations of cAMP. The kinase was found to inactivate Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk), a heterodimer of Cdk5 and neuronal Cdk5 activator (Nck5a), under phosphorylation conditions. The phosphorylation of Nclk by the purified thymus kinase occurred on Cdk5. The monomeric form of Cdk5 was also phosphorylated by the kinase. Phosphoamino acid and phosphopeptide analysis of the phosphorylated Nclk revealed that Thr-14 of Cdk5 was the sole site of protein phosphorylation. The results suggest that this thymus kinase is a novel Cdk inhibitory protein kinase, distinct from the recently cloned dual functional and membrane-associated Cdc2 inhibitory kinase, Myt1 (Mueller, P. R., Coleman, T. R., Kumagai, A., and Durphy, W. G. (1995) Science 270, 86-90).
Insights
Researchers identified a novel protein kinase in bovine thymus that phosphorylates Thr-14. This kinase inhibits cyclin-dependent kinases (Cdks) like Cdk5, suggesting a new regulatory mechanism for cell cycle control.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Cyclin-dependent kinases (Cdks) regulate the cell cycle.
- Phosphorylation is a key mechanism for regulating protein activity.
- The specific kinases involved in Cdk regulation are not fully elucidated.
Purpose of the Study:
- To identify and characterize a novel protein kinase from bovine thymus cytosol.
- To investigate the substrate specificity and regulatory properties of this novel kinase.
- To determine if this kinase plays a role in regulating Cdk activity.
Main Methods:
- Synthetic peptide substrates (cdc2(6-20) and analogue) were used to assay kinase activity.
- Protein purification was performed based on peptide phosphorylation activity.
- SDS-PAGE and in-gel autophosphorylation were used to identify the kinase.
- Inhibition studies with cAMP were conducted.
- Assays with Cdc2/cyclin B, Cdk2/cyclin A, and neuronal Cdc2-like kinase (Nclk) were performed to assess kinase activity towards Cdks.
Main Results:
- A novel 43-kDa protein kinase was purified from bovine thymus cytosol.
- The kinase phosphorylated Thr-14 in synthetic peptides and in the Cdk5 subunit of Nclk.
- The kinase's activity was inhibited by millimolar concentrations of cAMP.
- The purified kinase inactivated Cdc2/cyclin B, Cdk2/cyclin A, and Nclk.
- Thr-14 of Cdk5 was identified as the sole phosphorylation site on Nclk.
Conclusions:
- A novel Cdk inhibitory protein kinase distinct from Myt1 was identified in bovine thymus.
- This kinase specifically phosphorylates Thr-14 on Cdk5, suggesting a novel regulatory pathway for Cdks.
- The findings contribute to understanding the complex regulation of cell cycle progression.