Activation of PKC Alpha by EGF and the Subsequent Phosphorylation of Tropomodulin

Abstract

Abstract: : 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.

Keywords: 383 cytoskeleton • 423 growth factors/growth factor receptors