Human Kinase                                                              Kinase Clone Distribution

Leonardo Brizuela, Ph.D.
Jaehong Park, Ph.D.
Dongmei Zuo, M.S.

Protein phosphorylation is a common post-translational modification of proteins and plays a pivotal role on protein structure and function. Due to its effects and its reversibility (due to the action of phosphatases), protein phosphorylation has a central role in all aspects of cell metabolism. Protein kinases play a central role in regulation of growth factor receptor activity, regulation of gene transcription, regulation of protein synthesis, control of ubiquitin-mediated protein degradation, as well as playing critical role in triggering fundamental cellular events such as mitosis and DNA synthesis. Finally, a large number of protein kinases have been involved in cancer and other large number of pathological conditions and diseases (The Protein Kinase Resource webpage, http://pkr.sdsc.edu/html/index.shtml).

Transfer of the gamma phosphate group from ATP or GTP to serine, threonine or tyrosine residues in proteins (via formation of phosphoester bonds) is catalyzed by protein kinases. The serine/threonine and the tyrosine kinase families represent two main families of this class of enzymes. The estimated number of proteins which are phosphorylated in the cell is high, with this number and its distribution changing significantly with respect to the physiological conditions of the cell in question. Furthermore, a large number of proteins contain multiple phosphorylation sites, and the consequences of the differential phosphorylation can be antagonist or additive in nature depending on the protein. This complexity dictates that not only the phospho-content of a protein to be identified but that the exact position of the phosphorylation to be unequivocally mapped. Detection of kinase activity has traditionally relied on the existence of radio labeled nucleotides and detection of the incorporated radio labeled phosphate into proteins substrates by means of X-ray radiography, liquid scintillation and phospho-imaging techniques. Newer techniques rely in the generation and use of phospho-specific antibodies, polarized fluorescence and mass spectrometry

Here at the Institute of Proteomics we are interested in cloning all the kinases encoded by the human genome. This gene set will also include kinases other than protein kinases, such as lipid-, sugar-, nucleotide- kinases, etc. In the context of the FLEXGene repository and in combination of the right cell based-assay, this gene set will represent an extraordinary tool that will facilitate the experimental approach to questions such as: what is the role of a given kinase under given physiologycal/pathologycal conditions of a cell , what is(are) the physiological substrate(s) of a given kinase?, and what are the consequences of the phosphorylation event(s) on a protein’s activity or function?.

Search of the NCBI databases (both protein and gene databases) suggest that the number of human kinases with complete sequence information is reaching approximately 1000 as of the summer of 2002. We have downloaded the sequence information of these genes and have generated a human kinase database. We are now in the process of cloning all these genes, in their wild type and dominant negative forms, using recombination-based cloning technologies. The immediate use of this gene set will be in the form of its effect on cell-based assays (by eliciting or inhibiting a giving phenotype). Some of the cell-based assays to be tested will include those with relevant read outs for sugar and insulin metabolism, growth factor/cytokine signaling pathways, apoptosis and other aspects of tumor biology, as well as immunosuppression. These functional genomic analyses will provide valuable information for elucidating the role of the different kinases in different cell types, and for the identification of targets for therapeutic intervention in various human pathologies.