title: Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo.
abstract: 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 PKA (cAMP-dependent protein kinase), Akt (protein kinase B), CLK2 (CDC-like kinase 2) and PKC (protein kinase C) alpha all phosphorylate CK1delta. PKA 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: PKA was detectable in the CK1deltaCK peak fraction of fractionated MiaPaCa-2 cell extracts, PKA shared nearly identical kinetic properties with those of CK1deltaCK, and both PKA and CK1deltaCK phosphorylated CK1delta at Ser370 in vitro. Furthermore, phosphorylation of CK1delta by PKA 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 PKA 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 PKA, resulted in a 6-fold increase in the activity of CK1delta. In summary, we conclude that PKA phosphorylates CK1delta, predominantly at Ser370 in vitro and in vivo, and that site-specific phosphorylation of CK1delta by PKA plays an important role in modulating CK1delta-dependent processes.
authors.affiliation: Clinic of General, Visceral and Transplantation Surgery, University of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany.
authors.forename: Georgios
authors.initials: G
authors.lastname: Giamas
authors.forename: Heidrun
authors.initials: H
authors.lastname: Hirner
authors.forename: Levani
authors.initials: L
authors.lastname: Shoshiashvili
authors.forename: Arnhild
authors.initials: A
authors.lastname: Grothey
authors.forename: Susanne
authors.initials: S
authors.lastname: Gessert
authors.forename: Michael
authors.initials: M
authors.lastname: Kühl
authors.forename: Doris
authors.initials: D
authors.lastname: Henne-Bruns
authors.forename: Constantinos E
authors.initials: CE
authors.lastname: Vorgias
authors.forename: Uwe
authors.initials: U
authors.lastname: Knippschild
mesh_terms: D000818:Animals; D015153:Blotting, Western; D048148:Casein Kinase Idelta / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D066298:In Vitro Techniques; D008845:Microinjections; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics / Q000378:metabolism*
chemical_list: D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D012694:Serine; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D048148:Casein Kinase Idelta; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C