Use of a novel method to find substrates of protein kinase C delta identifies M2 pyruvate kinase.
Protein kinase C (PKC) family members have been implicated in numerous cellular processes. However, identifying the substrates of each PKC isozyme remains a challenge. Here, we describe a method using two-dimensional (2D) isoelectric focusing gel electrophoresis to identify substrates of delta PKC (deltaPKC) in MCF-7 breast carcinoma cells. We show that M2 pyruvate kinase is a substrate of deltaPKC, and further characterize the interaction between M2 pyruvate kinase and deltaPKC in MCF-7 cells by immunoprecipitation. deltaPKC activation in vitro or in cells did not appear to alter the enzyme activity or polymerization of M2 pyruvate kinase.
Siwko
Stefan
S
Cancer Biology Program, CCSR Room 3145, 269 Campus Dr., Stanford University, Stanford, CA 94305, USA. siwko@bcm.edu
Mochly-Rosen
Daria
D
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007525:Isoelectric Focusing / Q000379:methods; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000737:chemistry / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D011770:Pyruvate Kinase; D051745:Protein Kinase C-delta