Abstract
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 Ser(8) and Thr(138) 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 Ser(91), 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 Ser(8), Ser(91), and Thr(138) phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser(8) was phosphorylated in all p35 molecules, whereas Ser(91) was phosphorylated in 60% and Thr(138) was phosphorylated in approximately 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.
Highlights
Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes
Cyclin-dependent kinase 5 (Cdk5) is activated by binding to activation subunit p35 and inactivated by proteasomal degradation of p35 [25]
Banding Profiles of Phosphorylated p35 in Phos-tag SDSPAGE—We first examined whether Phos-tag SDS-PAGE can be used to analyze the phosphorylation states of p35. p35 is phosphorylated at Ser8 by Cdk5 in adult rat brain [33]. p35 in postnatal day 1 (P1) rat brain extract was compared between Laemmli and Phos-tag SDS-PAGE after incubation with ATP and/or the phosphatase inhibitor Microcystin LR
Summary
Cyclin-dependent kinase; Phostag, phosphate-binding tag; NMDA, N-methyl-D-aspartate; BAP, bacterial alkaline phosphatase; CaMKII, Ca2ϩ-calmodulin-dependent protein kinase II; ERK, extracellular signal-regulated kinase; P, postnatal day; E, embryonic day; DIV, days in vitro; kn, kinase-negative; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Ca2ϩ-CaM, calcium and calmodulin. Phosphorylation of p35 Analyzed by Phos-tag SDS-PAGE postmitotic neurons and regulates various neuronal events such as neuronal migration, spine formation, synaptic activity, and cell death [22,23,24]. To understand the in vivo regulation of Cdk activity, it is critical to analyze the phosphorylation states of p35 in brain. We applied the Phos-tag SDS-PAGE method to analyze the phosphorylation states of p35 in vivo and in cultured neurons. We constructed standard band profiles of phosphorylated p35 by Phos-tag SDS-PAGE using Ala mutants at Ser and/or Thr138. From these experiments, we observed an unidentified in vivo phosphorylation site at Ser. We discuss the usefulness of Phos-tag SDS-PAGE to analyze the in vivo phosphorylation states of proteins
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