Quantitative measurement of in vivo phosphorylation states of Cdk5 activator p35 by Phos-tag SDS-PAGE

Tomohisa Hosokawa, Taro Saito, Akiko Asada, Kohji Fukunaga, Shin Ichi Hisanaga

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46 Citations (Scopus)


Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/ threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser91, which was phosphorylated by Ca 2+-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser8, Ser 91, and Thr138 phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser8 was phosphorylated in all p35 molecules, whereas Ser91 was phosphorylated in 60% and Thr138 was phosphorylated in ∼12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.

Original languageEnglish
Pages (from-to)1133-1143
Number of pages11
JournalMolecular and Cellular Proteomics
Issue number6
Publication statusPublished - 2010 Jun

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology


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