Cross Talk Between Protein Kinase CK2 And The Transcription Factor Pdx-1

Phosphorylation is one of the most important mechanisms for the post-translational modification of proteins. Studies of mammalian cells metabolically labeled with [32P] orthophosphate suggest that as many as one-third of all cellular proteins are covalently modified by protein phosphorylation.Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes, of particular importance is its involvement in signal transduction. The process of phosphorylation is under the control of protein kinases by transfering a phosphate group from ATP/GTP to the specific substrate protein.The human genome contains 518 protein kinase genes and they constitute about 2% of all human genes. As one of the first identified protein kinases, Casein kinase 2 is a highly conserved, ubiquitously expressed, serine/threonine protein kinase that is a heterotetramer (α2β2,α'2β2orαα'β2)composed of two catalytic subunits(αorα')and two regulatory subunits(β).CK2 has been implicated in various physiological and pathological cellular processes, such as gene expression, protein sythesis, cell cycle regulation as well as anti-apopotosis, carcinogenesis and etc. Until the year 2003, there are up to 307 proteins identified as CK2 substrates and more than one-fifth of these are implicated in gene expression as transcriptional factors.Pdx-1 (Pancreatic and duodenal homeobox-1) is a homeodomain transcription factor and functions as a masterfactor in the transcription of a large amount ofβ-cell specific genes including the insulin gene. Physiologically, Pdx-1 is necessary for pancreatic development and differentiation. In addition, it is also involved in the maintainance of adultβ-cell function. Pathologically, Pdx-1 gene mutations were closely related to the onset and development of diabetes.Furthermore, recent findings also revealed that Pdx-1 was overexpressed in various cancers including pancreatic cancer, colorectal cancer, lung cancer, breast cancer and etc.It was stated also in these findings that Pdx-1 was related to the survival of some of these cancer cells and thus supposed to be implicated in the carcinogenesis, development, metastasis and drug resistanse of cancers. It is thus crucial and a hot topic to determine the molecular mechanisms involved in the regulation of Pdx-1 expression and activation.There is increasing evidence showing that Pdx-1 is a phosphoprotein and that post-translational phosphorylation plays important roles in the regulation of some of its functions.In the present study we identified Pdx-1 as a bona fide substrate for CK2 by both in vitro phosphorylation assays and in vivo labelling of cells with phosphate, additional site-directed mutation analysis of Pdx-1 enabled us to find out the specific phosphorylation sites for CK2, namely Ser232 and Thr231,on the C-terminal region of Pdx-1 protein.Most interestingly, by employing luciferase reporter assay, cycloheximide(protein sythesis inhibitor) treatment and MG132(proteasome inhibitor) treatment experiments, we demonstrated that the phosphorylation of Pdx-1 by CK2 decreased its transcriptional activity on the insulin promoter, and on the other hand, CK2 phosphorylation also drived Pdx-1 to degradation which is probably in a proteasome dependent manner.Further immnofluroscence studies revealed that CK2 phosphorylation was not implicated in the regulation of the subcellular localization of Pdx-1. In contrast, overlay images showed a colocalization of Pdx-1 and individual CK2 subunits (α,α',β) in the nucleous, suggesting that there might be inter-molecular interactions between these proteins. Therefore, GST-pull down and Far-western blot assays as well as immunoprecipitation experiments were performed to identify the protein protein binding between Pdx-1 and CK2. We demonstrated that Pdx-1 directly interacted with both the CK2 holoenzyme and the individual subunits in vitro and were associated with all the three subunits of CK2 in vivo.In conclusion, we have shown in the present study the results of the existence of phosphorylation and interaction between CK2 and Pdx-1.Further functional analysis relating to the phosphorylation and interaction also provided a mechanism for temporal and spatial regulation of Pdx-1.