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Fig. 2. Kalirin-Thr1590 is phosphorylated by Cdk5. (A) GST and His-Myc-tagged proteins used in these studies. (B) Kalirin peptides were incubated with 4 ng recombinant Cdk5-p25 complex and 2 µCi [
32P]ATP for 30 minutes at 30°C; cpm incorporated into peptide is on the y-axis. Km and Vmax were determined by varying the concentration of peptide from 3.7-100 µM. (C) Recombinant GST-KGEF1-7end and 
Kalirin-7 are Cdk5-p25 substrates. Recombinant proteins (2 µg) were incubated without or with 4 ng recombinant Cdk5-p25 complex and 2 µCi [32P]ATP for 30 minutes at 30°C. Following fractionation by SDS-PAGE and transfer to a PVDF membrane, samples were visualized by autoradiography (16 hours) or by Coomassie Brilliant Blue staining; the first lane was loaded with molecular size markers. (D) Lysates of pEAK RAPID cells, PC12 cells and mouse striatum (20 µg) were fractionated by SDS-PAGE; endogenous Cdk5, p35, p39 and PP1 were visualized. (E) Top, synthetic Kalirin peptides were incubated with [32P]ATP in the presence of Cdk5-p35 complex immunoprecipitated from pEAK RAPID cells expressing exogenous Cdk5 and p35 using a p35 antibody; 10 µM roscovitine was included in the indicated assays (black bar). Bottom left, indicated doses of roscovitine were incubated with TPAK peptide. Bottom right, TPAK peptide was incubated with [32P]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-Kalirin-7 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 Kalirin-7 was visualized using a ThrPro-P antibody. Expression of DN Cdk5 eliminated the band detected by the ThrPro-P antibody.
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