Fig. 2. Kalirin-Thr1590 is phosphorylated by Cdk5.
A. GST and His/myc tagged proteins used in these studies are illustrated. B. The Kalirin peptides were incubated with 4ng recombinant Cdk5/p25 complex and 2μCi γ32P-ATP for 30min at 30C; cpm incorporated into peptide is on the y-axis. Km and Vmax were determined by varying the concentration of peptide from 3.7 to 100μM. C. Recombinant GST-KGEF1→7end and ΔKalirin7 are Cdk5/p25 substrates. Recombinant proteins (2μg) were incubated without or with 4ng recombinant Cdk5/p25 complex and 2μCi γ32P-ATP for 30min at 30C. Following fractionation by SDS-PAGE and transfer to a PVDF membrane, samples were visualized by autoradiography (16h) or by Coomassie Brilliant Blue staining; the first sample was loaded with the molecular weight marker. D. Lysates of pEAK RAPID cells, PC12 cells and mouse striatum (20μg) were fractionated by SDS-PAGE; endogenous Cdk5, p35, p39 and PP-1 were visualized. E. Top, synthetic Kalirin peptides were incubated with γ32P-ATP in the presence of Cdk5/p35 complex immunoprecipitated from pEAK RAPID cells expressing exogenous Cdk5 and p35 using a p35 antibody; 10μM roscovitine was included in the indicated assays (black bar). Middle, indicated doses of roscovitine were incubated with TPAK peptide. Bottom, TPAK peptide was incubated with γ32P-ATP in the presence of Cdk5/p35 or DN-Cdk5/p35 complex immunoprecipitated (p35 antibody) from pEAK RAPID cells. Incorporation of 32P into peptide was quantified by Cerenkov counting. F. pEAK RAPID cells expressing myc-Kalirin7 or parent vector (−) and DN-Cdk5 were extracted for immunoprecipitation using spectrin repeat region antibody. Inputs were analyzed using antibody to myc or Cdk5.
Immunoprecipitated Kalirin7 was visualized using a phospho-ThrPro antibody. Expression of DN-Cdk5 eliminated the band detected by the P-ThrPro antibody.