PNAS central -- Submit and Review Online Sign up for PNAS Online eTocs
Link: Info for AuthorsLink: Editorial BoardLink: AboutLink: SubscribeLink: AdvertiseLink: ContactLink: Sitemap Link: PNAS Home
Proceedings of the National Academy of Sciences
Link: Current Issue "" Link: Archives "" Link: Online Submission ""  Link: Advanced Search

Published online on May 31, 2005, 10.1073/pnas.0503141102
PNAS | June 7, 2005 | vol. 102 | no. 23 | 8114-8119


This Article
Right arrow Figures Only
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supporting Information
Right arrow Online Correction to Supporting Data Set 1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a colleague
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My File Cabinet
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, J.
Right arrow Articles by Brizuela, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, J.
Right arrow Articles by Brizuela, L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg  
What's this?

 Previous Article  | Table of Contents |  Next Article 

APPLIED BIOLOGICAL SCIENCES
Building a human kinase gene repository: Bioinformatics, molecular cloning, and functional validation

Jaehong Park *, Yanhui Hu *, T. V. S. Murthy *, Fredrik Vannberg *, Binghua Shen *, Andreas Rolfs *, Jessica E. Hutti {dagger}, Lewis C. Cantley {dagger}, Joshua LaBaer *, Ed Harlow *, and Leonardo Brizuela *, {ddagger}

*Harvard Institute of Proteomics, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 320 Charles Street, Cambridge, MA 02141; and {dagger}Department of Systems Biology, Harvard Medical School, 330 Brookline Avenue, Boston, MA 02115

Contributed by Ed Harlow, April 21, 2005

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.

kinome | autophosphorylation | cell-based screens | high-throughput cloning | survivin

Author contributions: J.P., Y.H., and L.B. designed research; J.P., Y.H., T.V.S.M., and J.E.H. performed research; J.P., B.S., A.R., L.C.C., J.L., E.H., and L.B. contributed new reagents/analytic tools; J.P., Y.H., T.V.S.M., F.V., and J.E.H. analyzed data; and J.P. and L.B. wrote the paper.

Abbreviations: HT, high throughput; MGC, Mammalian Gene Collection; shRNA, short-hairpin RNA; TCF, T cell factor.

Data Deposition: The gene constructs reported in this paper have been deposited in the GenBank database (accession nos. AY335555–AY335786).

{ddagger} To whom correspondence should be addressed. E-mail: lbrizuela{at}hms.harvard.edu.

© 2005 by The National Academy of Sciences of the USA


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg    What's this?


This article has been cited by other articles in HighWire Press-hosted journals:


Home page
 Proc. Natl. Acad. Sci. USAHome page
A. Rolfs, W. R. Montor, S. S. Yoon, Y. Hu, B. Bhullar, F. Kelley, S. McCarron, D. A. Jepson, B. Shen, E. Taycher, et al.
Production and sequence validation of a complete full length ORF collection for the pathogenic bacterium Vibrio cholerae
PNAS, March 18, 2008; 105(11): 4364 - 4369.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
D. Zuo, S. E. Mohr, Y. Hu, E. Taycher, A. Rolfs, J. Kramer, J. Williamson, and J. LaBaer
PlasmID: a centralized repository for plasmid clone information and distribution
Nucleic Acids Res., January 12, 2007; 35(suppl_1): D680 - D684.
[Abstract] [Full Text] [PDF]