The interaction and phosphorylation of tropomodulin by protein kinase Calpha in N/N 1003A lens epithelial cells.
OBJECTIVE
Tropomodulin, a tropomyosin and actin-binding protein stabilizes tropomyosin-actin filaments and is important in maintaining the elongated shape of lens fiber cells. In this study the role of PKCalpha-catalyzed phosphorylation of tropomodulin is determined.


METHODS
The interaction of PKCalpha and tropomodulin was measured by immunoprecipitation after activation with either phorbol ester at 200 nM for 60 min or 10 ng/ml EGF for 15 min. Tropomodulin phosphorylation was determined after co-immunoprecipitation using an in vitro [gamma-32P] PKC activity assay and by specific reaction with antiphosphothreonine antisera. Changes in tropomodulin interaction with tropomyosin or with the cytoskeleton were measured in a gel overlay assay and by association with a "Triton-insoluble" fraction.


RESULTS
Both phorbol ester and EGF caused an increased interaction of PKCalpha with tropomodulin. Following activation of PKCalpha by phorbol ester or by EGF there was an increased phosphorylation of tropomodulin on threonine residues. The phosphorylation of tropomodulin did not affect interaction with tropomyosin as measured by a gel overlay assay. However, there was an increased association of tropomodulin with the "Triton-insoluble" cytoskeletal fraction.


CONCLUSIONS
Activation of PKCalpha by EGF causes an increased phosphorylation of tropomodulin which results in an increase in tropomodulin association with cytoskeletal components. This establishes a signal pathway by which EGF induced activation of PKCalpha alters the interaction of lens cytoskeletal proteins.
Wagner
Lynn M
LM
Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA.
Fowler
Velia M
VM
Takemoto
Dolores J
DJ
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D007527:Isoenzymes / Q000502:physiology*; D007908:Lens, Crystalline / Q000166:cytology*; D008840:Microfilament Proteins / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D051571:Protein Kinase C-alpha; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014162:Transfection; D051348:Tropomodulin
D002352:Carrier Proteins; D007527:Isoenzymes; D008840:Microfilament Proteins; D051348:Tropomodulin; D013912:Threonine; D004815:Epidermal Growth Factor; C087857:protein kinase C gamma; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate