Phosphorylation of CK1delta: identification of Ser370 as the major phosphorylation site targeted by PKA in vitro and in vivo
Authors: Michael Kühl
Georgios Giamas
Levani Shoshiashvili
Heidrun Hirner
Susanne Gessert
Constantinos E Vorgias
Uwe Knippschild
Doris Henne-Bruns
Arnhild Grothey
Journal: Biochemical Journal (2007)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: CK1delta; phosphorylation; PKA; Akt; CLK2
Affiliations: Clinic of General, Visceral and Transplantation Surgery, University of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany., Clinic of General, Visceral and Transplantation Surgery, University of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany, Institute for Biochemistry and Molecular Biology, Albert-Einstein-Allee 11, 89081 Ulm, Germany, Department of Biochemistry and Molecular Biology, National and Kapodistrian University of Athens, Panepistimiopolis-Zographou, 15784 Athens, Greece

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. 

Retracted: No

PubPeer comments:
null, 
null, 
null, 
 It has come to the attention of the authors that in the original Figure 2(D) the same images were used for GST-CK1u03b41-428, GST-CK1u03b41-428S370A and GST-CK1u03b41-375 in the Coomassie-stained gel. In the autoradiogram of Figure 2(D), PKA negative GST-CK1u03b41-428, and PKA negative GST-CK1u03b41-428S370A were the same, as were GST-CK1u03b41-350 and GST-CK1u03b41-375. This mistake has now been addressed and corrected. In Figure 3(A) fraction number 14 of the control was accidentally used for fraction number 14 of H89, degeulin, and calphostin treatments. In addition, fraction number 13 of the control was erroneously used for fraction 13 in the deguelin treatment. Furthermore, fraction number 9 of the deguelin treatment was run on another gel, and the image was subsequently inserted into the Figure. The authors are now presenting the corrected Figures 2(D) and 3(A), and would like to apologize for any confusion that these errors may have caused. The new Figures have been assessed and approved by the Editorial Board of the Biochemical Journal. New Figure 2(D) ![file] New Figure 3(A) ![file] More available [here] . ,