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MARCKS protein is transcriptionally down-regulated in v-Src-transformed BALB/c 3T3 cells
C K Joseph1, S A Qureshi, D J Wallace
1Institute for Biomolecular Structure and Function, Hunter College, City University of New York, New York 10021.
Abstract:
Activation of protein kinase C (PKC) by tumor-promoting phorbol esters leads to the phosphorylation of an 80-kilodalton PKC substrate (known as MARCKS) in murine fibroblasts. In BALB/c 3T3 cells stably transformed by v-Src, phorbol esters were unable to induce phosphorylation of MARCKS. Western blot analysis and in vitro kinase assays showed that both PKC protein levels and kinase activity were unchanged in v-Src-transformed relative to the parental nontransformed BALB/c 3T3 cells. However, MARCKS protein levels were reduced in v-Src-transformed cells relative to nontransformed cells. MARCKS RNA levels were also correspondingly reduced in v-Src-transformed cells. Nuclear "run-on" assays showed decreased transcription of MARCKS in v-Src-transformed cells. Thus, the absence of MARCKS in v-Src-transformed cells could be explained by a down-regulation of MARCKS transcription. Inhibiting the protein tyrosine kinase activity of v-Src with herbimycin A restored MARCKS RNA levels, MARCKS transcription, and MARCKS protein, suggesting that down-regulation of MARCKS in v-Src-transformed BALB/c 3T3 cells is a direct effect of v-Src.
Insights
v-Src transformation in fibroblasts reduces the protein and RNA levels of MARCKS by down-regulating its transcription. This effect is reversed by inhibiting v-Src tyrosine kinase activity, indicating v-Src directly controls MARCKS expression.
Area of Science:
- Cell biology
- Molecular oncology
- Signal transduction
Background:
- Protein kinase C (PKC) activation by phorbol esters phosphorylates the 80-kDa substrate MARCKS in fibroblasts.
- The oncogene v-Src alters cellular signaling pathways and transformation.
Purpose of the Study:
- To investigate the effect of v-Src transformation on MARCKS phosphorylation and expression.
- To elucidate the mechanism underlying altered MARCKS levels in v-Src-transformed cells.
Main Methods:
- Western blot analysis to assess protein levels.
- In vitro kinase assays to measure kinase activity.
- Nuclear run-on assays to evaluate gene transcription.
- Treatment with herbimycin A to inhibit v-Src activity.
Main Results:
- v-Src-transformed cells failed to phosphorylate MARCKS upon phorbol ester stimulation.
- MARCKS protein and RNA levels were significantly reduced in v-Src-transformed cells.
- MARCKS transcription was decreased in v-Src-transformed cells, and this was restored by herbimycin A.
Conclusions:
- v-Src transformation leads to down-regulation of MARCKS expression at the transcriptional level.
- The protein tyrosine kinase activity of v-Src directly mediates the reduction in MARCKS transcription and protein levels.