Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus.
Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.
Freie Universität Berlin, Institut für Chemie-Biochemie, AG Neurochemie, Thielallee 63, 14195 Berlin, Germany.
Uwe
U
Rosenberger
Ingo
I
Lehmann
Christoph
C
Weise
Peter
P
Franke
Ferdinand
F
Hucho
Klaus
K
Buchner
D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000378:metabolism; D000072670:PTB-Associated Splicing Factor; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D009687:Nuclear Proteins; D000072670:PTB-Associated Splicing Factor; D016601:RNA-Binding Proteins; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha