The non-canonical functions of p27(Kip1) in normal and tumor biology

Abstract

p27(Kip1) was first discovered as a key regulator of cell proliferation. The canonical function of p27(Kip1) is inhibition of cyclin-dependent kinase (CDK) activity. In addition to its initial identification as a CDK inhibitor, p27(Kip1) has also emerged as an intrinsically unstructured, multifunctional protein with numerous non-canonical, CDK-independent functions that exert influence on key processes such as cell cycle regulation, cytoskeletal dynamics and cellular plasticity, cell migration, and stem-cell proliferation and differentiation. Many of these non-canonical functions, depending on the cell-specific contexts such as oncogenic activation of signaling pathways, have the ability to turn pro-oncogenic in nature and even contribute to tumor-aggressiveness and metastasis. This review discusses the various non-canonical, CDK-independent mechanisms by which p27(Kip1) functions either as a tumor-suppressor or tumor-promoter.

Keywords: cell cycle; cell migration; cyclin-dependent kinases; metastasis; non-canonical functions; p27Kip1; stem-cell biology.

Figure 1.

Linear representation of the key functional domains of p27 along with sites of post-translational modifications and protein-protein interactions. The p27 protein, encoded by CDKN1B, is 198-amino acids (aa) long in humans. The N-terminal portion of p27 harbors the cell-cycle inhibitory region that contains the cyclin- and CDK-binding domains comprising of aa 25–93 (shown in gray). The critical residues within this region required for binding to cyclins and CDKs are indicated. This region also harbors a nuclear export signal (NES), shown in red. The location of the nuclear localization signal (NLS) in the C-terminal portion is shown in green. The major sites of phosphorylation are denoted by black circles. The recently discovered sites of SUMOylation and acetylation are denoted by a star and open circle, respectively. The approximate sites of protein-protein interactions and other functional domains are denoted by brackets, below which each arrow points to the protein/microRNA partners of p27 interacting with that specific region (highlighted in blue); the interactions with CRM1 and mNPAP60, and post-translational modification by SUMOylation, included here for completeness, have not been discussed in the text.

Figure 2.

p27 regulates cell migration. Depending on the cell-specific context, p27 can either promote or inhibit cell migration in a CDK-independent manner. (a) RhoA functions as a molecular switch that cycles between inactive GDP-bound and active GTP-bound states that are promoted by GTPase-activating proteins (GAPs) and guanine–nucleotide exchange factors (GEFs), respectively. Upon stimulation, GEFs activate RhoA which in turn activates ROCKs to phosphorylate LIMKs. Thus activated, LIMK then phosphorylates cofilin to inhibit its actin-depolymerizing function to stabilize actin stress fibers and focal adhesions. Cytoplasmic p27 inhibits this pathway by directly interacting with RhoA and preventing its activation by the Rho-GEFs, resulting in increased cell motility. Mitogenic signaling pathways and oncogenic kinases target p27 for cytoplasmic relocalization through phosphorylation of specific residues (indicated by yellow circles)—phosphorylation of S10 promotes nuclear export while phosphorylation of T157 and/or T198 impairs nuclear import. (b) In certain cell types, cytoplasmic p27 can promote Rac activation to stimulate filopodium formation and cell migration (c) In migrating cortical interneurons, sustained activation of RhoA in the absence of p27 results in elevated actomyosin contractions due to increased phosphorylation of myosin-II light chain (pMLC-II) by activated MLC kinase (MLCK). This deregulation contributes to defective nucleokinesis, neurite branching, and tangential migration. Cytoplasmic p27 fine-tunes the actomyosin contractions by inhibiting RhoA activation to correct this defect. (d) p27 is a microtubule (MT)-associated protein and it promotes MT-polymerization, thereby contributing to neurite extension during interneuron migration. (e) p27 controls MT dynamics in a stathmin-dependent manner. Sequestration of stathmin, a MT-destabilizing protein, by p27 blocks the tubulin-sequestration activity of the former resulting in increased MT polymerization and inhibition of migration.

Figure 3.

A simplified schematic highlighting the non-canonical roles of p27 in stem-cell biology and metastasis. Nuclear p27 regulates differentiation in various cell types. p27 levels are low in mouse and human embryonic stem cells (mESCs and hESCs) and it markedly increases as these cells are induced to differentiate. (a) p27 contributes to transcriptional repression of SOX2, a pluripotency gene, during differentiation of pluripotent cells and in differentiated cells by associating with the SOX2-SRR2 enhancer together with the repressive complex p130-E2F4-SIN3A. (b) In differentiating pluripotent cells p27 associates with Brachyury and Twist1 promoters to repress the transcription of these genes; the identity of the repressor complex that p27 associates with on these promoters remains to be defined. (c) Neurogenin2 (Ngn2) is a proneural basic helix-loop-helix transcription factor that plays a central role in the neuronal differentiation of cortical progenitors. p27 plays a key role in this process by stabilizing the neurogenin2 protein in a CDK-independent manner. (d) Cytoplasmic p27 contributes to tumorigenesis and metastasis by exerting influence on various processes such as expansion of the stem cell pool, cell migration and invasion, and epithelial-mesenchymal transition (see text for details).

Figure 4.

p27 in cell-cycle regulation. In addition to inhibition of cyclin-CDK activities, p27 also contributes to regulation of the cell-cycle by non-canonical means. (a) p27 is a RNA-binding protein. In contact-inhibited cells, cytoplasmic p27 directly binds and stabilizes miR-223. High levels of miR-223 then target E2F1 mRNA and thereby promote cell cycle arrest. (b) p27 also contributes to G0-G1 arrest by functioning as a transcriptional co-repressor in a CDK-independent fashion. On the promotors of specific target-genes it associates with p130/E2F4 complex and facilitates gene repression by recruiting co-repressors such as HDAC1 and mSIN3A. (c and d) p27 controls activation of MAPK pathway in a CDK-independent manner to regulate cell cycle entry. (c) In mitogen-stimulated quiescent cells, p27 is rapidly exported to the cytoplasm where it binds free GRB2 to limit GRB2-SOS complex formation and thus has the potential to attenuate MAPK activation. (d) In mitogen-stimulated cells, p27 sequesters stathmin to counteract its microtubule-destabilizing activity. The resulting enhanced microtubule-stability facilitates increased endocytic-trafficking of H-Ras and its ubiquitination, leading to attenuation of H-Ras –MAPK signaling and inhibition of cell cycle entry. (e) p27 interacts in a CDK-independent fashion with MCM7, a key component of the pre-replication complex that is assembled at the origins of replication. This interaction might prevent premature firing of the origins in late G1. (f) p27 sequesters cyclin F in a CDK-independent manner and antagonizes the cyclin F-mediated degradation of CP110 resulting in centrosome amplification and mitotic catastrophe. (g) Citron kinase (Citron-K) is essential for cytokinesis; p27 interferes with normal cytokinesis by interacting with citron-K and preventing its activation by RhoA.