Differential regulation of protein kinase C isoforms byv-Src.
Item
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Title
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Differential regulation of protein kinase C isoforms byv-Src.
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Identifier
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AAI9720156
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identifier
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9720156
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Creator
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Zang, Qun.
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Contributor
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Adviser: David A. Foster
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Cell | Biology, Molecular
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Abstract
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The protein kinase C (PKC) family consists of at least 11 distinct isoforms. In both murine and rat fibroblasts, we detected expression of the conventional PKC {dollar}\alpha{dollar}, the novel PKCs {dollar}\delta{dollar} and {dollar}\varepsilon{dollar}, and the atypical PKC {dollar}\zeta{dollar}. Except for the atypical PKCs, membrane association has been used to determine PKC activation. In cells transformed by v-Src, there was a Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent increase in membrane association of the {dollar}\alpha{dollar} isoform relative to the non-transformed parental cells. Of the novel PKC isoforms {dollar}\delta{dollar} and {dollar}\varepsilon{dollar}, the {dollar}\delta{dollar} isoform was preferentially associated with the membrane in v-Src-transformed cells. Since it is not clear whether the subcellular distribution of a PKCs correlates with their activation, we could not determine whether {dollar}\zeta{dollar} isoform is activated. PKC {dollar}\delta{dollar} and PKC {dollar}\varepsilon{dollar} are both activated by exogenous diacylglycerol and phorbol ester. Thus, the differential activation of the {dollar}\delta{dollar} and {dollar}\varepsilon{dollar} isoforms by v-Src suggests that the regulation of the novel PKC isoforms involves more complex mechanism.;We found that PKC {dollar}\delta{dollar} co-immunoprecipitates with v-Src and is phosphorylated on tyrosine. The tyrosine phosphorylated PKC {dollar}\delta{dollar} was primarily localized in membrane fraction. However, tyrosine-phosphorylated PKC {dollar}\delta{dollar} has reduced enzymatic activity relative to non-tyrosine-phosphorylated PKC {dollar}\delta{dollar}. c-Src did not co-immunoprecipitate with PKC {dollar}\delta{dollar} and an activated c-Src mutant (c-Src 527F) did, suggesting that the association between Src and PKC {dollar}\delta{dollar} requires active Src kinase. An additional mutation at the N-terminal of c-Src 527F, abolishing membrane association of Src, prevented its association with and tyrosine phosphorylation of PKC {dollar}\delta{dollar}. A deletion within the SH2 domain of Src did not prevent the Src/PKC {dollar}\delta{dollar} interaction. Interestingly, both the association between c-Src-527F and PKC {dollar}\delta{dollar} and the tyrosine phosphorylation of PKC {dollar}\delta{dollar} were substantially enhanced by mutating the PKC phosphorylation site at Ser 12 in Src, suggesting that phosphorylation of Src by PKC destabilizes the interaction in a negative feedback loop. These data suggest a complex regulation of PKC {dollar}\delta{dollar} isoform in which direct interaction between Src and PKC {dollar}\delta{dollar} may result in PKC {dollar}\delta{dollar} tyrosine phosphorylation and down-regulation of its kinase activity.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
