Mechanism of cell cycle entry mediated by the intrinsically disordered protein p27(Kip1)

p27(Kip1) (p27), a prototypical intrinsically disordered protein (IDP), regulates eukaryotic cell division through interactions with cyclin-dependent kinase (Cdk)/cyclin complexes. The activity, stability, and subcellular localization of p27 are regulated by phosphorylation. We illustrate how p27 integrates regulatory signals from several non-receptor tyrosine kinases (NRTKs) to activate Cdk4 and initiate cell cycle entry. Unmodified p27 potently inhibits Cdk/cyclin complexes, including Cdk4/cyclin D (IC(50), 1 nM). Some NRTKs (e.g., Abl) phosphorylate p27 on Tyr 88, which facilitates a second modification on Tyr 74 by another NRTK (e.g., Src). Importantly, this second modification causes partial reactivation of Cdk4 within ternary complexes containing doubly Tyr phosphorylated p27. Partial activation of Cdk4 initiates entry into the cell division cycle. Therefore, p27's disordered features enable NRTKs to sequentially promote a phosphorylation cascade that controls cell fate. Beyond cell cycle control, these results illustrate general concepts regarding why IDPs are well-suited for roles in signaling and regulation in biological systems.     

Tyrosine phosphatase SHP-2 is a regulator of p27Kip1 tyrosine phosphorylation

Tyrosine phosphorylation of the cell cycle regulator p27^Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27^Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27^Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27^Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27^Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27^Kip1 stability and contributes to p27^Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27^Kip1 tyrosine phosphorylation.     

Incomplete folding upon binding mediates Cdk4/cyclin D complex activation by tyrosine phosphorylation of inhibitor p27 protein

p27(Kip1) (p27), an intrinsically disordered protein, regulates the various Cdk/cyclin complexes that control cell cycle progression. The kinase inhibitory domain of p27 contains a cyclin-binding subdomain (D1), a Cdk-binding subdomain (D2), and a linker helix subdomain that connects D1 and D2. Here, we report that, despite extensive sequence conservation between Cdk4/cyclin D1 (hereafter Cdk4/cyclin D) and Cdk2/cyclin A, the thermodynamic details describing how the individual p27 subdomains contribute to equally high affinity binding to these two Cdk/cyclin complexes are strikingly different. Differences in enthalpy/entropy compensation revealed that the D2 subdomain of p27 folds incompletely when binding Cdk4/cyclin D versus Cdk2/cyclin A. Incomplete binding-induced folding exposes tyrosine 88 of p27 for phosphorylation by the nonreceptor tyrosine kinase Abl. Importantly, tyrosine phosphorylation (of p27) relieves Cdk inhibition by p27, enabling cell cycle entry. Furthermore, the interaction between a conserved hydrophobic patch on cyclin D and subdomain D1 is much weaker than that with cyclin A; consequently, a construct containing subdomains D1 and LH (p27-D1LH) does not inhibit substrate binding to Cdk4/cyclin D as it does to Cdk2/cyclin A. Our results provide a mechanism by which Cdk4 (within the p27/Cdk4/cyclin D complex) is poised to be activated by extrinsic mitogenic signals that impinge upon p27 at the earliest stage of cell division. More broadly, our results further illustrate the regulatory versatility of intrinsically disordered proteins.     

p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2

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.     

Direct cell cycle regulation by the fibroblast growth factor receptor (FGFR) kinase through phosphorylation-dependent release of Cks1 from FGFR substrate 2

Fibroblast growth factors (FGFs) are upstream activators of the mitogen-activated protein kinase pathway and mitogens in a wide variety of cells. However, whether the mitogen-activated protein kinase pathway solely accounts for the induction of cell cycle or antiapoptotic activity of the FGF receptor (FGFR) tyrosine kinase is not clear. Here we report that cell cycle inducer Cks1, which triggers ubiquitination and degradation of p27(Kip1), associates with the unphosphorylated form of FGFR substrate 2 (FRS2), an adaptor protein that is phosphorylated by FGFR kinases and recruits downstream signaling molecules. FGF-dependent activation of FGFR tyrosine kinases induces FRS2 phosphorylation, causes release of Cks1 from FRS2, and promotes degradation of p27(Kip1) in 3T3 cells. Since degradation of p27(Kip1) is a key regulatory step in activation of the cyclin E/A-Cdk complex during the G(1)/S transition of the cell cycle, the results suggest a novel mitogenic pathway whereby FGF and other growth factors that activate FRS2 directly activate cyclin-dependent kinases.     

P27 expression is regulated by separate signaling pathways, downstream of Ras, in each cell cycle phase

The cyclin inhibitory protein p27Kip1 (p27) plays a vital role in regulating cell proliferation in response to the extracellular growth environment. Active proliferation requires the suppression of p27 levels throughout the cell cycle. Late in the cell cycle, p27 degradation requires phosphorylation of Thr 187 by cyclin dependent kinase 2, leading to recognition by the SCF ubiquitin ligase containing the Skp2 F-box protein. Suppression of p27 is also essential for cell proliferation early in the cell cycle, but this occurs independently of Skp2, whose expression is suppressed during G1 phase. In this study, we use a time lapse and quantitative imaging approach to study the connection between proliferative signaling and the degradation of p27 during each cell cycle period in actively cycling cells. Ras activity was required for the suppression of p27 levels throughout the cell cycle, but separate pathways downstream of Ras signaling were required in different cell cycle periods. For example, inhibitors of MEK and phosphatidylinositol-3-kinase induced p27 expression primarily in G1 phase, while inhibitors of AKT activity stimulated these levels primarily in S phase. Skp2 was expressed in a Ras-dependent manner at higher levels late in the cell cycle. Its ablation resulted in higher p27 levels primarily in G2 phase as expected. The fact that separate signaling pathways downstream of Ras function in each cell cycle phase to suppress p27 levels helps explain the vital connection between proliferative signaling, cell cycle control, and p27 expression.     

Cdk-inhibitory activity and stability of p27Kip1 are directly regulated by oncogenic tyrosine kinases

p27Kip1 controls cell proliferation by binding to and regulating the activity of cyclin-dependent kinases (Cdks). Here we show that Cdk inhibition and p27 stability are regulated through direct phosphorylation by tyrosine kinases. A conserved tyrosine residue (Y88) in the Cdk-binding domain of p27 can be phosphorylated by the Src-family kinase Lyn and the oncogene product BCR-ABL. Y88 phosphorylation does not prevent p27 binding to cyclin A/Cdk2. Instead, it causes phosphorylated Y88 and the entire inhibitory 3(10)-helix of p27 to be ejected from the Cdk2 active site, thus restoring partial Cdk activity. Importantly, this allows Y88-phosphorylated p27 to be efficiently phosphorylated on threonine 187 by Cdk2 which in turn promotes its SCF-Skp2-dependent degradation. This direct link between transforming tyrosine kinases and p27 may provide an explanation for Cdk kinase activities observed in p27 complexes and for premature p27 elimination in cells that have been transformed by activated tyrosine kinases.     

Growth, differentiation, and malignant transformation of pre-B cells mediated by inducible activation of v-Abl oncogene

The nonreceptor tyrosine kinase, encoded by the v-Abl oncogene of Abelson murine leukemia virus induces transformation of progenitor B cells. The v-Abl oncogene promotes cell cycle progression and inhibits pre-B cell differentiation. The temperature-sensitive form of Abelson murine leukemia virus offers a reversible model to study the role of v-Abl in regulating growth and differentiation. Inactivation of v-Abl elevates p27 and Foxo3a levels and activates NF-kappaB/Rel, which leads to G1 arrest and induction of Ig L chain gene rearrangement, respectively. In turn, v-Abl reactivation reduces p27 and Foxo3a levels, thus permitting G1-arrested cells to reenter the cell cycle. However, the cell lines derived from SCID mice that are defective in the catalytic subunit of DNA-dependent protein kinase retain elevated levels of p27 and Foxo3a proteins despite reactivation of v-Abl. Consequently, these cells are locked in the G1 phase for an extended period of time. The few cells that manage to bypass the G1 arrest become tumorigenic and fail to undergo pre-B cell differentiation induced by v-Abl inactivation. Deregulation of p27, Foxo3a, c-myc, and NF-kappaB/Rel was found to be associated with the malignant transformation of SCID temperature-sensitive form of Abelson murine leukemia virus pre-B cells.     

A cyclin-abundance cycle-independent p34cdc2 tyrosine phosphorylation cycle in early sea urchin embryos.

The activity of the cell cycle control protein p34cdc2 is post-translationally regulated in a variety of cell types. Using anti-phosphotyrosine antibodies, we find that p34cdc2-directed tyrosine kinase activity increases at fertilization in sea urchin eggs, leading to a gradual accumulation of phosphotyrosine on p34 during the early part of the cell cycle. Loss of phosphotyrosine from p34 accompanies entry into mitosis and phosphotyrosine reaccumulates as the embryo enters the next cell cycle. A similar pattern is seen when eggs are parthenogenetically activated with ammonium chloride. Tyrosine phosphorylation and phosphorylation/dephosphorylation cycles are suppressed when embryos are treated with the tyrosine kinase inhibitor genistein. On the other hand, a cycle persists when protein synthesis is inhibited with emetine, indicating that it is independent of the synthesis of another class of cell cycle control proteins, the cyclins. Additional experiments with the phorbol ester, phorbol myristate acetate, demonstrate that activating protein synthesis alone in unfertilized eggs does not result in stimulation of p34cdc2 tyrosine kinase activity. Our results indicate that p34 tyrosine phosphorylation cycles are triggered by the fertilization Cai transient. The first cycle is independent of the fertilization pHi signal, confirming that, in sea urchin embryos, the cycle is not tightly coupled to the cycle of cyclin abundance that is a prominent feature of the eukaryotic cell division cycle.     

Tumor suppressor gene p16/INK4A/CDKN2A‐dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer

p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16‐defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16‐transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re‐feeding to induce cell cycle re‐entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16‐associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re‐entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by ³H‐thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16‐transfected CMT27A and CMT27H cells exited cell cycle post‐serum‐starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post‐serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non‐proliferative states. J. Cell. Biochem. 114: 1355–1363, 2013. © 2012 Wiley Periodicals, Inc.     

p18INK4c and p27KIP1 are required for cell cycle arrest of differentiated myotubes

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27(-/-) or p18(-/-)p27(-/-) myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18(-/-)p27(-/-) myotubes than in p27(-/-) myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18(-/-)p27(-/-) myotube proliferation to encompass approximately half of the nuclei. p18(-/-)p21(-/-) and p21(-/-)p27(-/-) myotubes remain terminally arrested. Nuclei within individual restimulated p18(-/-)p27(-/-) myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18(-/-)p27(-/-) myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18(-/-)p27(-/-) myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18(-/-)p27(-/-) myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.     

Constitutive MEK/MAPK activation leads to p27(Kip1) deregulation and antiestrogen resistance in human breast cancer cells

Antiestrogens, such as the drug tamoxifen, inhibit breast cancer growth by inducing cell cycle arrest. Antiestrogens require action of the cell cycle inhibitor p27(Kip1) to mediate G1 arrest in estrogen receptor-positive breast cancer cells. We report that constitutive activation of the mitogen-activated protein kinase (MAPK) pathway alters p27 phosphorylation, reduces p27 protein levels, reduces the cdk2 inhibitory activity of the remaining p27, and contributes to antiestrogen resistance. In two antiestrogen-resistant cell lines that showed increased MAPK activation, inhibition of the MAPK kinase (MEK) by addition of U0126 changed p27 phosphorylation and restored p27 inhibitory function and sensitivity to antiestrogens. Using antisense p27 oligonucleotides, we demonstrated that this restoration of antiestrogen-mediated cell cycle arrest required p27 function. These data suggest that oncogene-mediated MAPK activation, frequently observed in human breast cancers, contributes to antiestrogen resistance through p27 deregulation.     

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.     

Role of intrinsic flexibility in signal transduction mediated by the cell cycle regulator, p27 Kip1

p27^Kip1 (p27), which controls eukaryotic cell division through interactions with cyclin-dependent kinases (Cdks), integrates and transduces promitogenic signals from various nonreceptor tyrosine kinases by orchestrating its own phosphorylation, ubiquitination and degradation. Intrinsic flexibility allows p27 to act as a “conduit” for sequential signaling mediated by tyrosine and threonine phosphorylation and ubiquitination. While the structural features of the Cdk/cyclin-binding domain of p27 are understood, how the C-terminal regulatory domain coordinates multistep signaling leading to p27 degradation is poorly understood. We show that the 100-residue p27 C-terminal domain is extended and flexible when p27 is bound to Cdk2/cyclin A. We propose that the intrinsic flexibility of p27 provides a molecular basis for the sequential signal transduction conduit that regulates p27 degradation and cell division. Other intrinsically unstructured proteins possessing multiple sites of posttranslational modification may participate in similar signaling conduits.     

Rat protein tyrosine phosphatase eta suppresses the neoplastic phenotype of retrovirally transformed thyroid cells through the stabilization of p27(Kip1)

The r-PTPeta gene encodes a rat receptor-type protein tyrosine phosphatase whose expression is negatively regulated by neoplastic cell transformation. Here we first demonstrate a dramatic reduction in DEP-1/HPTPeta (the human homolog of r-PTPeta) expression in a panel of human thyroid carcinomas. Subsequently, we show that the reexpression of the r-PTPeta gene in highly malignant rat thyroid cells transformed by retroviruses carrying the v-mos and v-ras-Ki oncogenes suppresses their malignant phenotype. Cell cycle analysis demonstrated that r-PTPeta caused G(1) growth arrest and increased the cyclin-dependent kinase inhibitor p27(Kip1) protein level by reducing the proteasome-dependent degradation rate. We propose that the r-PTPeta tumor suppressor activity is mediated by p27(Kip1) protein stabilization, because suppression of p27(Kip1) protein synthesis using p27-specific antisense oligonucleotides blocked the growth-inhibitory effect induced by r-PTPeta. Furthermore, we provide evidence that in v-mos- or v-ras-Ki-transformed thyroid cells, the p27(Kip1) protein level was regulated by the mitogen-activated protein (MAP) kinase pathway and that r-PTPeta regulated p27(Kip1) stability by preventing v-mos- or v-ras-Ki-induced MAP kinase activation.