Correlation between protein kinase C binding proteins and substrates in REF52 cells.
We have used a blot overlay assay to identify phosphatidylserine-dependent interactions between protein kinase C (PKC) and PKC binding proteins. The purpose of the present studies was to compare the properties of PKC binding proteins and PKC substrates detected by in vivo and in vitro phosphorylation assays. The major binding proteins and substrates in REF52 cells shared similar properties including enrichment by calmodulin-Sepharose chromatography, binding to phosphatidylserine, and resistance to heat denaturation. In addition, several of the major binding proteins and substrates were coordinately down modulated in SV40-transformed REF52 cells. The major PKC substrate, MARCKS, was also detected as a PKC binding protein. These results emphasize that the phosphatidylserine-dependent interactions between PKC and several substrates are of sufficient affinity to be detected in a blot overlay. Down modulation of binding proteins/substrates in transformed cells may reflect either decreased expression or increased basal phosphorylation of the target proteins and is likely to be involved in maintenance of the transformed phenotype.
Hyatt
S L
SL
W. Alton Jones Cell Science Center, Lake Placid, New York 12946.
Liao
L
L
Aderem
A
A
Nairn
A C
AC
Jaken
S
S
D015153:Blotting, Western / Q000379:methods; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D015536:Down-Regulation; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D008970:Molecular Weight; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011489:Protein Denaturation; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D002148:Calmodulin-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D010718:Phosphatidylserines; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha