Cyclin D and cyclin-dependent kinase 4 (cdk4) are overexpressed in a variety of tumors, but their levels are not accurate indicators of oncogenic activity because an accessory factor such as p27Kip1 is required to assemble this unstable dimer. Additionally, tyrosine (Y) phosphorylation of p27 (pY88) is required to activate cdk4, acting as an "on/off switch." We identified two SH3 recruitment domains within p27 that modulate pY88, thereby modulating cdk4 activity. Via an SH3-PXXP interaction screen, we identified Brk (breast tumor-related kinase) as a high-affinity p27 kinase. Modulation of Brk in breast cancer cells modulates pY88 and increases resistance to the cdk4 inhibitor PD 0332991. An alternatively spliced form of Brk (Alt Brk) which contains its SH3 domain blocks pY88 and acts as an endogenous cdk4 inhibitor, identifying a potentially targetable regulatory region within p27. Brk is overexpressed in 60% of breast carcinomas, suggesting that this facilitates cell cycle progression by modulating cdk4 through p27 Y phosphorylation. p27 has been considered a tumor suppressor, but our data strengthen the idea that it should also be considered an oncoprotein, responsible for cyclin D-cdk4 activity.C yclin D1-cyclin-dependent kinase 4 (cdk4) complexes promote the G 0 /G 1 -phase transition, and as such their activity is tightly regulated by a variety of mechanisms, including the transcription and translation of the mitogen sensor cyclin D1 and positive and negative regulatory phosphorylation of cdk4 (1, 2). The best-characterized substrate of cyclin D-cdk4 is the G 1 gatekeeper, retinoblastoma (Rb), and deregulation of cdk4 potentially accelerates Rb phosphorylation and cell cycle transitioning, promoting cancer development (3). Cyclin D1 and cdk4 are overexpressed in a variety of human cancers, and in mouse models, loss of either cdk4 or cyclin D1 prevents the development of certain oncogene-driven tumors, further evidence of their involvement (4-6). However, the levels of cyclin D or cdk4 in a tumor may not be reliable measures of activity, due to the fact that a third protein, an assembly factor such as p27Kip1 or p21Cip1, is required both for the stabilization and then the subsequent activation of this complex (1, 7).Independently of its ability to assemble cyclin D-cdk4 complexes, p27 acts as a bona fide "switch" turning cyclin D-cdk4 complexes on or off, which in turn modulates cell cycle entry or exit (8, 9). Tyrosine (Y) phosphorylation of p27 on residues Y74, Y88, and Y89 opens the cyclin D-cdk4-p27 ternary complex, rendering it able to phosphorylate substrates such as Rb (9-14). Cyclin D-cdk4-p27 complexes isolated from cells in G 0 lack Y phosphorylation on p27 and are catalytically inactive, while complexes isolated from proliferating cells are Y phosphorylated and active. Y88 and Y89 are part of the 3-to-10 helix, which has been shown to insert into the cdk ATP binding cleft (15). When not phosphorylated, residues Y88 and Y89 (Y88/Y89) sequester within this binding pocket and block cdk4 activity (p2...
The oncogenes Cyclin D and cdk4 are overexpressed in a variety of tumors, but the levels of these proteins are not always accurate indicators of oncogenic activity because p27Kip1 is required to assemble this otherwise unstable dimer. However, p27’s association activates or alternatively inhibits cyclin D-cdk4, serving as a bona fide ON/OFF “switch.” Tyrosine (Y) phosphorylation of residues Y88/89 in p27 displaces its C-terminus from the cdk4 active site, permitting both ATP binding and CAK phosphorylation of cdk4’s T loop. This model leads to the following hypothesis: modulation of p27 Y phosphorylation controls cdk4 activity, which in turn regulates efficient cell cycle passsage, and in cancers where cdk4 activity is deregulated, p27 may be constitutively switched ON. Deregulated Src Family Kinase (SFK) signaling in cancer may increase p27 Y phosphorylation, constitutively activating oncogenic cdk4, causing continuous cell cycling. Using our p27 Y88 phosphospecific antibody, we have shown in primary tumors, that p27 Y phosphorylation is not detected in benign tissue regions, but is detected in grade 1 and progressively higher grade tumors, suggesting that p27 Y phosphorylation may be a marker for increased oncogenic cdk4 activity and cdk4 inhibitor sensitivity. Although SFKs have been implicated in p27 Y phosphorylation, little is known about the domains involved on either the SFK or p27. We identified two SH3 recruitment domains within p27 that modulate Y88 phosphorylation, thereby modulating cdk4 activity. Mutation of these domains results in loss of Y88 phosphorylation, while the prior addition of an SH3 peptide is able to prevent Y88 phosphorylation. Using a phage-ELISA assay, we identified PTK6/Brk, (Protein Tyrosine Kinase 6/Breast Tumor Kinase), that functions as a high-affinity kinase, able to phosphorylate p27 in vitro and associate with phosphorylated p27 in vivo. Overexpression of PTK6 in vivo increases Y88 phosphorylation and increases resistance to specific cdk4 inhibition by the chemical inhibitor, PD0332991, in a kinase-dependent fashion. As PTK6/Brk is overexpressed in more than 60% of human breast carcinomas, our data suggest that PTK6/Brk overexpression facilitates cell cycle progression by increasing cdk4 activity through direct p27 Y phosphorylation. As PD0332991 moves into the clinic, p27 Y phosphorylation could serve as a marker to identify tumors sensitive to cdk4 inhibition, while blocking the PTK6:p27 interaction represents a novel therapeutic option to inhibit cdk4 activation. Citation Format: Stacy W. Blain, Cindy Gomez, Elina Shteyn, Priyank Patel, Susan R.S. Gottesman, Benedikt Asbach, Ralf Wagner, Angela L. Tyner. PTK6/BRK modulates tyrosine phosphorylation of p27Kip1 and the activity of the oncogene cyclin D-cdk4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-123. doi:10.1158/1538-7445.AM2013-LB-123
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