Identification of the smooth muscle-specific protein, sm22, as a novel protein kinase C substrate using two-dimensional gel electrophoresis and mass spectrometry.
We report a novel method to identify protein kinase C (PKC) substrates. Tissue lysates were fractionated by ion exchange chromatography and used as substrates in in vitro kinase reactions. The phosphorylated proteins were separated using two-dimensional gel electrophoresis. Spots that contained isolated phosphoproteins were excised and digested with trypsin. The tryptic peptides were analyzed using mass spectrometry. While several of the proteins identified using this technique represent known PKC substrates, we identified a new PKC substrate in the initial screen. This protein, sm22, is expressed in smooth muscle cells and served well as a substrate for PKC in vitro. Sm22 is predominantly associated with the actin cytoskeleton. Upon activation of PKC in vivo, sm22 dissociates from the actin cytoskeleton and is distributed diffusely in the cytoplasm. Our data strongly suggest that phosphorylation by PKC controls the intracellular localization of sm22. This demonstrates that our approach, using a complex mixture of proteins as in vitro kinase substrates and subsequently identifying the newly phosphorylated proteins by mass spectrometry, is a powerful method to identify new kinase substrates.
Institut für Klinische Molekularbiologie, GSF München, Germany. sascha.dammeier@byk.de
S
S
Dammeier
J
J
Lovric
M
M
Eulitz
W
W
Kolch
J F
JF
Mushinski
H
H
Mischak
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D007424:Intracellular Fluid / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008840:Microfilament Proteins; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013154:Spleen / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate
D008840:Microfilament Proteins; D009124:Muscle Proteins; C058992:Tagln protein, mouse; C051615:transgelin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate