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...