RESUMO
CK2 is a multifunctional, pleiotropic protein kinase involved in the regulation of cell proliferation and survival. Since fibroblasts from Type 1 Diabetes patients (T1DM) with Nephropathy exhibit increased proliferation, we studied cell viability, basal CK2 expression and activity, and response to specific CK2 inhibitors TBB (4,5,6,7-tetrabenzotriazole) and CX4945, in fibroblasts from T1DM patients either with (T1DM+) or without (T1DM-) Nephropathy, and from healthy controls (N). We tested expression and phosphorylation of CK2-specific molecular targets. In untreated fibroblasts from T1DM+, the cell viability was higher than in both N and T1DM-. CK2 inhibitors significantly reduced cell viability in all groups, but more promptly and with a larger effect in T1DM+. Differences in CK2-dependent phosphorylation sites were detected. In conclusion, our results unveil a higher dependence of T1DM+ cells on CK2 for their survival, despite a similar expression and a lower activity of this kinase compared with those of normal cells.
Assuntos
Caseína Quinase II/antagonistas & inibidores , Nefropatias Diabéticas/metabolismo , Fibroblastos/metabolismo , Adulto , Caseína Quinase II/metabolismo , Sobrevivência Celular , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Naftiridinas/farmacologia , Fenazinas , Inibidores de Proteínas Quinases/farmacologiaRESUMO
The ability of three isoforms of protein kinase CK1 (alpha, gamma(1), and delta) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1-28 sequence. Both substrates are readily phosphoylated by CK1delta and CK1alpha, but not by the gamma isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K (m) 0.82 muM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K(221)RQK(224) loop according to modeling and mutational analysis.
