Brk/Protein Tyrosine Kinase 6 Phosphorylates p27^KIP1, Regulating the Activity of Cyclin D–Cyclin-Dependent Kinase 4

Abstract

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...

Mechanisms of the CDK4/6 inhibitor palbociclib (PD 0332991) and its future application in cancer treatment (Review)

“…During interphase, the G1-S transition is a critical restriction point, resulting in one of three fates for the cell: continue cycling, exit active proliferation, or enter a quiescent (G0) state. Many growth factors and inhibitors interact to coordinate cell cycle progression, and the CDK4/6-Rb pathway (5,6) plays a central role in regulating the G1 to S phase transition. So far, 21 cyclin-dependent kinases (CDKs) have been identified in mammalian cells by the HUGO Gene Nomenclature Committee (HGNC) and the Mouse Genomic Nomenclature Committee (7).…”

Tracing the footprints of the breast cancer oncogene BRK — Past till present

“…5). An [78]. This is suggestive of another potential mecha-641 nism by which BRK contributes towards mammary oncogenesis [78].…”

“…Lately, p27Kip1 was reported as another novel substrate of BRK[78]. It was shown that p27Kip is recruited to the BRK SH3 domain and is phosphorylated on Y74, Y88 and Y89, which was suggested as a mechanism via which BRK promotes cell cycle progression[78].In a relatively recent discovery, heat shock protein 90 (Hsp90) was established as the major protein responsible for regulating endogenous BRK stability in the T47D and BT474 breast carcinoma cell lines[60].…”

Mechanisms of Resistance to CDK4/6 Inhibitors in Breast Cancer and Potential Biomarkers of Response

“…Other suggested mechanisms of de novo resistance to CDK4/6 inhibitors, involving deregulation of cell cycle-related proteins, include phosphorylation status of p27 and fizzy-related protein homolog (FZR1) [29,30]. In addition to its ability to inhibit cyclin D/CDK4, p27 can regulate the cyclin D/CDK4/p27 ternary complex activity depending on phosphorylation status of tyrosine Y74, Y88, and Y89, [31].…”

“…In addition to its ability to inhibit cyclin D/CDK4, p27 can regulate the cyclin D/CDK4/p27 ternary complex activity depending on phosphorylation status of tyrosine Y74, Y88, and Y89, [31]. It was shown that overexpression of Brk (breast tumor-related kinase), an intracellular tyrosine kinase overexpressed in 60% of breast cancers [32], increases phosphorylation on Y88 of p27 and cyclin D/CDK4 activity, rendering breast cancer cells more resistant to palbociclib [29]. …”