Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo.

The involvement of CK1 (casein kinase 1) delta in the regulation of multiple cellular processes implies a tight regulation of its activity on many different levels. At the protein level, reversible phosphorylation plays an important role in modulating the activity of CK1delta. In the present study, we show that (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and (protein kinase C) alpha all phosphorylate CK1delta. duanyu1529 was identified as the major cellular CK1deltaCK (CK1delta C-terminal-targeted protein kinase) for the phosphorylation of CK1delta in vitro and in vivo. This was implied by the following evidence: duanyu1529 was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, duanyu1529 shared nearly identical kinetic properties with those of CK1deltaCK, and both duanyu1529 and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by duanyu1529 decreased substrate phosphorylation of CK1delta in vitro. Mutation of Ser370 to alanine increased the phosphorylation affinity of CK1delta for beta-casein and the GST (gluthatione S-transferase)-p53 1-64 fusion protein in vitro and enhanced the formation of an ectopic dorsal axis during Xenopus laevis development. Anchoring of duanyu1529 and CK1delta to centrosomes was mediated by AKAP (A-kinase-anchoring protein) 450. Interestingly, pre-incubation of MiaPaCa-2 cells with the synthetic peptide St-Ht31, which prevents binding between AKAP450 and the regulatory subunit RII of resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that duanyu1529 phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by duanyu1529 plays an important role in modulating CK1delta-dependent processes.

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