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Activated Src abrogates the Myc requirement for the G0/G1 transition but not for the G1/S transition

Tulsiram Prathapam et al. Proc Natl Acad Sci U S A. 2006.

Abstract

The transcription factor Myc plays a central role in the control of cellular proliferation. Myc expression is induced by growth factors in a pathway mediated by cellular Src (c-Src), but it is not clear whether Myc induction or activity is required for malignant transformation by activated Src. We introduced v-Src into a c-myc(-/-) derivative of Rat-1 fibroblasts and into 3T9 mouse fibroblasts harboring a conditionally excisable c-myc allele. Expression of activated viral Src in Myc-deficient cells led to loss of actin stress fibers and surface fibronectin, indicating that Myc is dispensable for v-Src-induced morphological transformation. However, v-Src failed to rescue the proliferative defect resulting from the loss of Myc. In Myc-deficient cells, despite its inability to overcome this proliferation block, v-Src was able to regulate the expression of certain Myc transcriptional targets and induce the expression of active cyclin D/Cdk4 and Cdk6 complexes; it also induced the phosphorylation of Rb, albeit at reduced levels. In contrast, however, in the absence of Myc, the level of Cdk2 kinase activity was drastically reduced. This reduction in Cdk2 activity was associated with a decrease in the expression of Cdk7, Cdc25A, and cyclin A. Coexpression of Cdk2 plus cyclin E and/or cyclin A rescued the G1/S block and allowed the cells to enter mitosis. These results indicate that in the absence of Myc, v-Src can activate early G1 cell cycle regulators but fails to activate regulators of the late G1/S transition.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Effects of Myc deficiency on transformation of Rat1 and 3T9 cells by v-Src. (A) Morphological transformation of Myc-deficient Rat1 cells by v-Src. (A Left) Lysates of Rat1 myc+/+ and myc_−/− cells expressing either empty vector or v-Src were resolved by SDS/PAGE and immunoblotted with anti-Src mAb 2–17 or 4G10. (A Right) Phase contrast micrographs of same cells expressing either empty vector or v-Src. (B) Schematic showing the generation of Myc-deficient 3T9 cells by Cre-mediated myc excision. (C) Absence of Myc expression does not affect Src levels and activity. 3T9_mycflox/flox cells stably expressing Cre-ER and either empty vector or v-Src were induced with 4-OH-T to excise c-myc. Total RNA isolated from uninduced (Myc+) and 4-OH-T-induced (Myc−) cells was analyzed for myc mRNA levels by RT-PCR. Expression of v-Src and phosphotyrosine activity were analyzed by immunoblotting. (D) Expression of v-Src does not overcome the proliferative defect resulting from the deletion of c-myc. Equal numbers of 3T9_mycflox/flox_ cells stably expressing Cre-ER and either empty vector or v-Src were seeded in triplicate in medium containing 10% serum and induced with 4-OH-T for the indicated periods. Cells were trypsinized and counted with a Coulter counter. The data represent the number of cells relative to the number of cells at the time of plating (mean of three independent experiments). (E) v-Src expression does not rescue the defect in DNA synthesis resulting from the absence of Myc. Equal numbers of 3T9_mycflox/flox_ cells stably expressing Cre-ER and either empty vector or v-Src were induced with 4-OH-T for the indicated periods, and BrdUrd incorporation was assayed to determine the percentage of cells synthesizing DNA. Data represent the mean of three independent experiments ± SD. Myc levels were analyzed by RT-PCR.

Fig. 2.

Effects of Myc deficiency on the expression of Myc targets. Total RNA isolated from uninduced and 4-OH-T induced cells was analyzed by agarose gel electrophoresis and Northern hybridization, and RNA loading was monitored either by visualizing ribosomal RNAs or by Northern hybridization with a probe for GAPDH. Cell lysates were resolved by SDS/PAGE and protein expression monitored by immunoblotting, with either tubulin or GAPDH as loading control. (A) Expression of Gas1, Gadd45, p27, and cyclin D1. (B) Expression of nM23A and p21. (C) Expression of ODC and AIRC. (D) Expression of Cdk4 and Cdk6.

Fig. 3.

Effects of Myc deficiency on activation of cell cycle regulators by v-Src. (A) In the absence of Myc, v-Src induces cyclin D-dependent kinases but fails to activate Cdk2. In the top four blots, complexes were immunoprecipitated from extracts with antibodies indicated and kinase activities were assayed by using carboxyterminal Rb as substrate. In blots 5–7, cell lysates were analyzed by immunoblotting with anti-Rb antibody and phospho-specific Rb antibodies: _p_-T821 and _p_-S807 plus S811. (B) Failure of v-Src to induce active Cdk2 in c-_myc_−/− cells results from reduced expression of Cdk2, cyclin A, Cdk7, and Cdc25A. Equal amounts of lysates were analyzed by immunoblotting with antibodies directed against phospho T160-Cdk2, Cdk2, cyclins A and E, Cdk7, and Cdc25A. Cdk-activating enzyme/Cdk7 activity was measured by an immune complex kinase assay by using anti-Cdk7 antibody with recombinant Cdk2 protein as substrate. (C) The fraction of Cdk2 complexed to p21 is increased in the absence of Myc. p21 was immunodepleted from cell lysates by two successive rounds of immunoprecipitation with anti-p21 antibody, and coimmunoprecipitation of Cdk2 was monitored by immunoblotting. The supernatants obtained from the second round of p21 immunoprecipitation were again immunoprecipitated with anti-Cdk2 antibody to determine the levels of Cdk2 not bound to p21 (Bottom).

Fig. 4.

Cooverexpression of cyclins E and A and Cdk2 in v-Src transformed cells is able to rescue the G1/S block resulting from lack of Myc. 3T9_mycflox/flox_ Cre-ER v-Src-positive cells constitutively overexpressing Cdk2 were generated and then transiently transfected with expression plasmids as indicated: cyclin E, or cyclin A, both cyclin E and A, or cyclin E and a stabilized mutant of cyclin A (A47). Cells were induced with 4-OH-T to excise c-myc and BrdUrd incorporation was assayed to determine the percentage of cells synthesizing DNA. A and B represent two separate sets of experiments; for each set, the data represent the mean of three independent experiments ± SE. (A Lower) Cdk2 activity assayed as described in Fig. 3_A_. (B Lower) Immunoblotting with anti-phospho-T160-Cdk2 antibody. (C) Percentage of mitotic cells, obtained by anti-phospho-histone H3 staining.

Fig. 5.

Schematic model depicting the significance of Myc in v-Src transformation. In the absence of Myc, v-Src regulates early cell cycle regulators such as p27 and cyclin D-Cdk4 but fails to induce the G1/S transition because of a failure to activate cyclin E/Cdk2 and cyclin A/Cdk complexes.

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Activated Src abrogates the Myc requirement for the G0/G1 transition but not for the G1/S transition - PubMed (original) (raw)