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LM Wagner, VM Fowler, DJ Takemoto; Activation of PKC Alpha by EGF and the Subsequent Phosphorylation of Tropomodulin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2349.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if EGF activates PKC α and, upon activation, if PKC α phosphorylates tropomodulin. Methods: Ten nanograms per milliliter EGF was added to cells in DMEM for 15 minutes. Cell lysates were harvested in 50 mM Tris, pH 7.5 and 20 mM MgCl2 and centrifuged at 100,000 xg for 1 hour at 4ºC. The pellet and supernatant fractions were run on a SDS-PAGE gel and the separated protein was transferred onto a nitrocellulose membrane. The membrane was probed with anti-PKC α antibody to determine translocation of the enzyme to the plasma membrane (activation). Co-immunoprecipitations were performed to determine if there was an increased interaction as a result of EGF activation of PKC α. Two approaches were used to detect phosphorylation of tropomodulin in the immunoprecipitates from N/N 1003A cells. First, an in vitro PKC phosphorylation assay was done to determine if tropomodulin is phosphorylated by PKC α. Second, in order to determine if tropomodulin is phosphorylated on a threonine or serine residue, tropomodulin was immunoprecipitated and the transferred protein on a nitrocellulose membrane was probed with either anti-phosphothreonine or anti-phosphoserine antibody. We used Triton X-100 insolubility as an empirical measurement of tropomodulin association with the the cytoskeleton. Cell lysates were centrifuged at 13,000xg for 1 hour. The pellet and supernatant fractions were run on a SDS-PAGE gel and the separated protein was transferred onto a nitrocellulose membrane. The membrane was probed with anti-tropomodulin antibody. Results: EGF acts as an activator of PKC α which is measured by the translocation of the enzyme from the cytosol to the plasma membrane. Because EGF activates PKC α, this causes an increase in the interaction between PKC α and tropomodulin. Activated PKC α phosphorylates tropomodulin on threonine residue(s). This phosphorylation of tropomodulin did result in an increase in the proportion of tropomodulin associated with Triton-insoluble cytoskeleton. Conclusion: PKC α, when activated by EGF, phosphorylates tropomodulin on threonine residue(s). This phosphorylation leads to an increase in the association of tropomodulin to the cytoskeleton.
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