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Molecular cloning and sequencing of the cytostatic G protein-activated protein kinase PAK I
R Jakobi1, C J Chen, P T Tuazon
1Department of Biochemistry, University of California, Riverside 92521, USA.
Abstract:
The serine/threonine protein kinase PAK I (p2l-activated protein kinase), a ubiquitous multipotential protein kinase of 58-60 kDa, has been shown to have cytostatic properties. Data from our laboratory show that PAK I is highly active in oocytes and quiescent and serum-starved cells, and injection of active PAK I into one blastomere of two-cell frog embryos inhibits cleavage of the injected blastomere. To clone the cDNA encoding PAK I, purified peptides from rabbit PAK I were sequenced, degenerate oligonucleotides were used to isolate PAK I clones from a rabbit spleen library, and the 5'-terminus was obtained by polymerase chain reaction. The entire cDNA sequence extends over 4471 nucleotides, with an open reading frame for a protein of 524 residues and a 3'-noncoding region of 2826 nucleotides. Clones with the same open reading frame but with 3'-noncoding regions of 1055 and 2478 nucleotides were isolated, suggesting the generation of different transcripts by alternative termination of transcription. The amino acid sequence of PAK I shows high homology to the p2l-activated protein kinases from human placenta and rat brain and to yeast STE20. PAK I is activated by Cdc42(GTP). The PAK enzymes have been proposed to regulate the stress-activated protein kinase (also known as the Jun kinase) signaling pathway (Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146; Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157).
Insights
The serine/threonine protein kinase PAK I has cytostatic properties and is highly active in oocytes and quiescent cells. Its cDNA was cloned, revealing a 524-residue protein homologous to other PAKs and yeast STE20, activated by Cdc42(GTP).
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Serine/threonine protein kinase PAK I (p21-activated protein kinase) is a ubiquitous kinase with known cytostatic properties.
- PAK I exhibits high activity in oocytes and quiescent, serum-starved cells.
- Injection of active PAK I into frog embryos inhibits blastomere cleavage, suggesting a role in cell cycle regulation.
Purpose of the Study:
- To clone the cDNA encoding the serine/threonine protein kinase PAK I.
- To characterize the PAK I protein sequence and its relationship to other known PAKs.
- To investigate the activation mechanism and potential signaling pathways involving PAK I.
Main Methods:
- Peptide sequencing of purified rabbit PAK I.
- Degenerate oligonucleotide-based cloning from a rabbit spleen library.
- 5'-terminus amplification using polymerase chain reaction (PCR).
- cDNA sequencing and analysis of open reading frame and non-coding regions.
- Homology analysis with known protein kinases.
Main Results:
- The complete cDNA sequence of PAK I was determined (4471 nucleotides), encoding a 524-residue protein.
- Alternative transcription termination generated transcripts with different 3'-noncoding regions (2826, 1055, and 2478 nucleotides).
- PAK I amino acid sequence showed high homology to human and rat PAKs and yeast STE20.
- PAK I is activated by Cdc42(GTP).
Conclusions:
- The cloning and sequencing of PAK I provide a molecular basis for its function.
- PAK I's homology to other PAKs and STE20 suggests conserved roles in signaling pathways.
- PAK I activation by Cdc42(GTP) implicates it in regulating pathways like the stress-activated protein kinase (Jun kinase) pathway.