Cdk5-Dependent Regulation of Neuronal MEK1
INTRODUCTION: Cyclin-dependent protein kinase 5 (Cdk5) is a member of the Cdk family that is implicated in many regulatory pathways in post-mitotic neurons. The kinase plays an important role in neuronal development and synaptic transmission; its disregulation contributes to neurodegenerative diseases such as Alzheimer's disease. Cdk5 is involved in the regulation of the Ras-Raf-MEK-ERK signaling pathway. It is hypothesized that Cdk5 serves a neuroprotective role by preventing the prolonged stimulation of ERK from inducing cell cycle reentry and, hence, neuronal apoptosis. In order to address whether Cdk5 phosphorylation of MEK1 affects its in vitro kinase activity, we sought to confirm the Cdk5-phosphorylation site of MEK1. Although it has been previously shown that Cdk5 phosphorylates MEK1-T286, our previous unpublished mass spectometry analysis of in vitro kinase reactions identifies the site as T292. We corroborated this data with Western blot analysis of the in vitro Cdk5 phosphorylation of MEK with phosphate-specific antibodies to sites T286 and T292. METHODS: Glutathione S-transferase (GST)-tagged MEK1 was expressed and purified from E.coli. The tagged protein was run through column fractions, with glutathione beads that would attach to the GST tag, such that GST-MEK1 would be purified out of the bacterial lysates. This was then run through gel electrophoresis to confirm that MEK1 had indeed been purified, by comparing the bands seen in the gels to the known molecular weights of GST-MEK1. The protein concentration was determined using a Bradford assay. Non-radioactive and radioactive Cdk5 kinase assays of MEK1 were then performed. MEK1 was incubated in the absence or presence of Cdk5 with Mg2+/ATP or Mg2+/ATP plus trace amounts of [gamma-32P]-ATP, respectively. The non-radiolabeled kinase reactions were subjected to polyacrylamide gel electrophoresis (PAGE) on a 10-20% acrylamide gradient gel, followed by electrophoretic transfer and Western blot analysis with phospho-T286 and phospho-T292 MEK1 antibodies. Phosphorimages of radiolabeled kinase reactions were generated by PAGE, dried gels and standards were exposed to a phosphorimager screen and analyzed on a phosphorimager. RESULTS: Our results confirm that T292 is indeed the Cdk5 site of MEK1. The identity of this site is important because it is the location at which control can be exercised over the ERK pathway.