Building a human kinase gene repository: bioinformatics, molecular cloning, and functional validation.
Kinases catalyze the phosphorylation of proteins, lipids, sugars, nucleosides, and other important cellular metabolites and play key regulatory roles in all aspects of eukaryotic cell physiology. Here, we describe the mining of public databases to collect the sequence information of all identified human kinase genes and the cloning of the corresponding ORFs. We identified 663 genes, 511 encoding protein kinases, and 152 encoding nonprotein kinases. We describe the successful cloning and sequence verification of 270 of these genes. Subcloning of this gene set in mammalian expression vectors and their use in high-throughput cell-based screens allowed the validation of the clones at the level of expression and the identification of previously uncharacterized modulators of the survivin promoter. Moreover, expressions of the kinase genes in bacteria, followed by autophosphorylation assays, identified 21 protein kinases that showed autocatalytic activity. The work described here will facilitate the functional assaying of this important gene family in phenotypic screens and their use in biochemical and structural studies.
Park
Jaehong
J
Harvard Institute of Proteomics, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 320 Charles Street, Cambridge, MA 02141, USA.
Hu
Yanhui
Y
Murthy
T V S
TV
Vannberg
Fredrik
F
Shen
Binghua
B
Rolfs
Andreas
A
Hutti
Jessica E
JE
Cantley
Lewis C
LC
Labaer
Joshua
J
Harlow
Ed
E
Brizuela
Leonardo
L
D000818:Animals; D001681:Biological Assay; D002384:Catalysis; D002460:Cell Line; D002477:Cells / Q000378:metabolism; D003001:Cloning, Molecular; D019295:Computational Biology; D030541:Databases, Genetic; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D010641:Phenotype; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results
D011994:Recombinant Proteins; D011494:Protein Kinases