p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2

Abstract

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.

Figure 1. Src preferentially phosphorylates p27 at Y74 and Y88 in vitro

(A) Wt His-p27 reacted with Src kinase with or without PP1. (B) His-p27WT and p27 mutants reacted with Src kinase per Exp. Procedures. Radioactivity in autophosphorylated Src (pSrc) or p27 (³²P-His-p27) quantitated by phosphoimager, and His-p27 input are shown. (C)Quantitation of triplicate Src kinase assays (means +/− SEM). (D)His-p27WT and p27 mutants reacted with Yes kinase per Exp. Procedures. Radioactivity in Yes (pYes) or p27 (³²P-His-p27) quantitated by phosphoimager, and His-p27 input are shown. (E) Quantitation of triplicate Yes kinase assays (means +/− SEM).

Figure 2. Phosphorylation by Src reduces p27 inhibitory action on cyclin E-Cdk2

(A)His-p27WT was phosphorylated in vitro with activated Src. Mock treated (p27) or Src treated pY-p27 were incubated with cyclin E-Cdk2 and H1 kinase activity assayed. Equal input of mock vs. Src treated p27 shown. (B)His-p27WT incubated without Src (no Src), with inactive Src (dead Src) and active Src are shown. pYp27 was precipitated with αpY-4G10. Equal amounts of p27 from (B) were incubated with cyclin E-Cdk2. (C) p27, (D) Cdk2 and (E) Cyclin E were precipitated and associated proteins blotted.

Figure 3. pY74p27 antibody and in vivo p27 phosphorylation by Src

(A) His-p27WT or His-p27Y74F were reacted with Src in vitro. p27 precipitates were blotted with αpY-4G10 or αp27pY74 (left). MCF-7 was transfected with YFPp27WT and empty vector (Src −) or PCI-Src-Y530F (Src +). p27 precipitates were blotted with αpY-4G10, stripped and re-probed for p27 (middle panel). pY74p27 detected in Src transfected MCF-7 (right panel). (B) MCF-7 was co-transfected with YFPp27WT or mutant p27 with or without Src. pYp27 was detected in p27 precipitates with αpY-4G10 or αp27pY74. (C) Cyclin E, Cdk2 and YFPp27 levels in YFPp27WT transfected MCF-7 with or without Src cotransfection or PP1. p27 (D) and Cdk2 (E) precipitates were resolved and proteins detected by blotting.

Figure 4. cSrc inactivation increases p27 levels and stability and Src induction decreases p27 t_1/2

(A)MCF-7 cells transfected with cSrc siRNA or non-specific controls analyzed for cSrc, 27 and β-actin. (B)cSrc, p27 and β-actin levels with (+) or without (−) AZD0530 in MDA-MB-361. (C)Cycloheximide (CHX) chase of p27 in MDA-MB-361 before and 6h after AZD0530. (D) Src was induced in estrogen starved MCF-pINDSrc2 (E −) by ponasterone A (PA+). 6h after estrogen addition (E+), Src, p27 and β-actin were assayed. (E) CHX chase of p27 at 6 h after estrogen treatment, with or without prior Src induction. (F) p27pT187, p27 and β-actin at 13 h after E stimulation, with or without prior Src induction. MG132 added 6 h prior to analysis.

Figure 5. Src activation and p27-Src interaction increase in early G1

(A) p27, cSrc and β-actin, IP Src kinase activities and p27-bound cSrc in MCF-7, T-47D and MDA-MB-361. (B) cSrc, p27, p27-bound cSrc and nuclear (RCC1), cytoplasmic (tubulin) and membrane (caveolin) markers in subcellular fractions from quiescent (E−) or asynchronous (E+) T-47D. (C) Estrogen-deprived MCF-7 (t=0) were stimulated with estradiol and insulin and %S and protein levels assayed at indicated times. (D) cSrc activity, p27-bound cSrc, cyclin E and Cdk2 in lysates from (C) above. (E) Src, p27 and β-actin in estrogen starved cells treated with estrogen for 2 h with (+PA) or without (−PA) prior Src induction (left). p27 and Src in αp27pY74 precipitates (right).

Figure 6. Src inhibitor cooperates with tamoxifen to arrest antiestrogen resistant breast cancer cells

(A) Cell cycle profile in MDA-MB-361 treated with TAM, AZD0530 or both for 48 h. (B) Cyclin E, Cdk2 and p27 in cells from (A). (C) Cyclin E bound Cdk2 and p27.