Cytosolic and nuclear localization of protein phosphatase 2C beta 1 in COS and BHK cells.
We have recently elucidated the structure of an isoform of the Mg(2+)-dependent protein phosphatase 2C (PP2C beta 1) from rat liver (Wenk, J., H.-I. Trompeter, K.-G. Pettrich, P. T. W. Cohen, D. G. Campbell, G. Mieskes, FEBS Lett. 297, 135-138 (1992)). In the present study the subcellular distribution of PP2C beta 1 was investigated in COS and BHK cells. We modified the PP2C beta 1 cDNA with a c-myc tag coding sequence at either its 3' or its 5' end end to distinguish the plasmid derived PP2C beta 1 from the endogenous phosphatase and to examine the influence of the modification on the enzymatic activity. Transient transfections of COS or BHK cells with pCMV2 derived vectors containing these constructs showed that the expressed hybrid protein with the lowest activity (N-terminal tagged < untagged < C-terminal tagged PP2C beta 1) was expressed to the highest level and vice versa. These experiments point to a possible toxic effect or a selection disadvantage after overexpression of PP2C beta 1. In immunofluorescence studies with antibodies specific for the PP2C beta isoform, all overexpressed proteins showed the expected cytoplasmic as well as a significant nuclear localization. The nuclei remained stained even after selective perforation of the plasma membrane with digitonin and washing out the cytosolic PP2C beta 1. We conclude that PP2C beta 1 has obligatory and important functions in metabolic as well as in nuclear processes.
Wenk
J
J
Department of Clinical Biochemistry, University of Göttingen, Germany.
Mieskes
G
G
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D006224:Cricetinae; D003600:Cytosol / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D029701:Saccharomyces cerevisiae Proteins; C087358:PTC1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D000071636:Protein Phosphatase 2C