Giamas Georgios, Hirner Heidrun, Shoshiashvili Levani, Grothey Arnhild, Gessert Susanne, Kühl Michael, Henne-Bruns Doris, Vorgias Constantinos E, Knippschild Uwe
Clinic of General, Visceral and Transplantation Surgery, University of Ulm, Steinhoevelstr. 9, 89075 Ulm, Germany.
Biochem J. 2007 Sep 15;406(3):389-98. doi: 10.1042/BJ20070091.
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.
酪蛋白激酶1（CK1）δ参与多种细胞过程的调控，这意味着其活性在许多不同层面受到严格调控。在蛋白质水平上，可逆磷酸化在调节CK1δ的活性中起着重要作用。在本研究中，我们发现蛋白激酶A（PKA）、蛋白激酶B（Akt）、类细胞周期蛋白依赖性激酶2（CLK2）和蛋白激酶C（PKC）α均能使CK1δ磷酸化。在体外和体内，PKA被确定为使CK1δ磷酸化的主要细胞CK1δCK（CK1δ C末端靶向蛋白激酶）。以下证据表明了这一点：在分级分离的MiaPaCa - 2细胞提取物的CK1δCK峰组分中可检测到PKA，PKA与CK1δCK具有几乎相同的动力学特性，并且PKA和CK1δCK在体外均能使CK1δ的丝氨酸370磷酸化。此外，PKA使CK1δ磷酸化会降低CK1δ在体外的底物磷酸化水平。将丝氨酸370突变为丙氨酸会增加CK1δ在体外对β - 酪蛋白和谷胱甘肽S - 转移酶（GST）-p53 1 - 64融合蛋白的磷酸化亲和力，并在非洲爪蟾发育过程中增强异位背轴的形成。PKA和CK1δ定位于中心体是由A激酶锚定蛋白（AKAP）450介导的。有趣的是，用合成肽St - Ht31预孵育MiaPaCa - 2细胞，该肽可阻止AKAP450与PKA调节亚基RII之间的结合，导致CK1δ的活性增加6倍。总之，我们得出结论，PKA使CK1δ磷酸化，在体外和体内主要发生在丝氨酸370位点，并且PKA对CK1δ的位点特异性磷酸化在调节CK1δ依赖性过程中起重要作用。
