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A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle
Y Ben-David1, K Letwin, L Tannock
1Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Abstract:
In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.
Insights
Researchers discovered a novel class of protein kinases, named Clk, with dual tyrosine and serine/threonine kinase activity. These kinases may play a role in regulating cell cycle progression.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Protein kinases are crucial regulators of cellular processes.
- Tyrosine kinases and serine/threonine kinases have distinct roles in cell signaling and cycle control.
- Identifying novel kinases can reveal new regulatory pathways.
Purpose of the Study:
- To identify novel protein-tyrosine kinase coding sequences.
- To characterize the enzymatic activities of newly identified kinase candidates.
- To investigate the potential role of these kinases in cell cycle regulation.
Main Methods:
- Screening of mouse erythroleukemia cDNA expression libraries using anti-phosphotyrosine antibodies.
- Expression and purification of the Clk protein in bacteria.
- In vitro kinase assays using exogenous substrates and autophosphorylation.
- Biochemical characterization of kinase activities.
Main Results:
- Identified cDNAs encoding Clk and Nek proteins, related to cell cycle regulators but possessing kinase motifs.
- The Clk protein exhibits both protein-tyrosine and protein-serine/threonine kinase activities.
- Direct biochemical evidence confirmed intrinsic dual kinase activity within the Clk catalytic domain.
- Clk phosphorylates tyrosine residues on an exogenous substrate.
Conclusions:
- The Clk gene product represents a novel class of protein kinases.
- This kinase class possesses an unusual dual substrate specificity (tyrosine and serine/threonine).
- Clk kinases may be involved in the intricate regulation of cell cycle progression.