p32 (gC1qBP) is a general protein kinase C (PKC)-binding protein; interaction and cellular localization of P32-PKC complexes in ray hepatocytes.
The aim of this study was to identify cellular proteins that bind protein kinase C (PKC) and may influence its activity and its localization. A 32-kDa PKC-binding protein was purified to homogeneity from the Triton X-100-insoluble fraction obtained from hepatocytes homogenates. The protein was identified by NH(2)-terminal amino acid sequencing as the previously described mature form of p32 (gC1qR). Recombinant p32 was expressed as a glutathione S-transferase fusion protein, affinity-purified, and tested for an in vitro interaction with PKC using an overlay assay approach. All PKC isoforms expressed in rat hepatocytes interacted in vitro with p32, but the binding dependence on PKC activators was different for each one. Whereas PKCdelta only binds to p32 in the presence of PKC activators, PKCzeta and PKCalpha increase their binding when they are in the activated form. Other PKC isoforms such as beta, epsilon, and theta bind equally well to p32 regardless of the presence of PKC activators, and PKCmu binds even better in their absence. It was also found that p32 is not a substrate for any of the PKC isoforms tested, but interestingly, its presence had a stimulatory effect (2-fold for PKCdelta) on PKC activity. We also observed in vivo interaction between PKC and p32 by immunofluorescence and confocal microscopy. A time course of phorbol ester treatment of cultured rat hepatocytes (C9 cells) showed that PKCtheta and p32 are constitutively associated in vivo, whereas PKCdelta activation is required for its association with p32. Our data also showed that phorbol ester treatment induces a transient translocation of p32 from the cytoplasm to the cell nucleus. Together, these findings suggest that p32 may be a regulator of PKC location and function.
Robles-Flores
Martha
M
Department of Biochemistry, Faculty of Medicine, and Institute of Cellular Physiology, Autonomous National University of Mexico, Mexico D.F. 04510. rmartha@servidor.unam.mx
Rendon-Huerta
Erika
E
Gonzalez-Aguilar
Hector
H
Mendoza-Hernandez
Guillermo
G
Islas
Socorro
S
Mendoza
Valentin
V
Ponce-Castaneda
M Veronica
MV
Gonzalez-Mariscal
Lorenza
L
Lopez-Casillas
Fernando
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D018960:Hyaluronan Receptors; D008297:Male; D008562:Membrane Glycoproteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D011951:Receptors, Complement / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
C490195:C1qbp protein, rat; D018076:DNA, Complementary; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; C032402:complement 1q receptor; D017830:Octoxynol; D005982:Glutathione Transferase; D011493:Protein Kinase C