CDK5/P35 PHOSPHORYLATION

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cyclin-dependent kinase 5 (cdk5) is a protein kinase that regulates neuronal structure in the central nervous system. It is active only when paired with a regulatory partner; one such partner is known as p35. When stressed, neurons are believed to cause calpain-mediated cleavage of p35 to a smaller form known as p25. Increased levels of p25 are associated with Alzheimer's disease and have also have been linked, along with increased cdk5 activity, to amyotrophic lateral sclerosis. We are exploring phosphorylation of the cdk5/p35 complex since the kinase appears to be regulated by its phosphorylation state. Incubation of kinase preparations in vitro with ATP results in the phosphorylation of at least two sites within p35 and seven sites within cdk5, as analyzed by mass spectrometry. The exact site of phosphorylation has been determined in some cases, while others are currently being mapped. We are also conducting experiments to determine whether autophosphorylation or phosphorylation due to another kinase co-purified with the cdk5/p35 complex is being observed.