p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti

Strict control of cellular proliferation is required to shape the complex structures of the developing embryo. The organ of Corti, the auditory neuroepithelium of the inner ear in mammals, consists of two types of terminally differentiated mechanosensory hair cells and at least four types of supporting cells arrayed precisely along the length of the spiral cochlea. In mice, the progenitors of greater than 80% of both hair cells and supporting cells undergo their terminal division between embryonic day 13 (E13) and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal. Here we report that the correct timing of cell cycle withdrawal in the developing organ of Corti requires p27(Kip1), a cyclin-dependent kinase inhibitor that functions as an inhibitor of cell cycle progression. p27(Kip1) expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the hair cells and supporting cells. In wild-type animals, p27(Kip1) expression is downregulated during subsequent hair cell differentiation, but it persists at high levels in differentiated supporting cells of the mature organ of Corti. In mice with a targeted deletion of the p27(Kip1) gene, proliferation of the sensory cell progenitors continues after E14, leading to the appearance of supernumerary hair cells and supporting cells. In the absence of p27(Kip1), mitotically active cells are still observed in the organ of Corti of postnatal day 6 animals, suggesting that the persistence of p27(Kip1) expression in mature supporting cells may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. Homozygous mutant mice are severely hearing impaired. Thus, p27(Kip1) provides a link between developmental control of cell proliferation and the morphological development of the inner ear.     

Confluence-induced cell cycle exit involves pre-mitotic CDK inhibition by p27Kip1 and cyclin D1 downregulation

Tissue homeostasis requires precise control of cell proliferation and arrest in response to environmental cues. In situation such as wound healing, injured cells are stimulated to divide, but as soon as confluence is reached proliferation must be blocked. Such reversible cell cycle exit occurs in G1, requires pRb family members, and is driven by p27Kip1-dependent Cdk inactivation. This implies that, while dividing, cells should simultaneously prepare the exit once mitosis is accomplished. For a long time, the decision to cycle or not was presumed to occur in G1, prior to the restriction point, beyond which the cells were bound to divide even in the absence of mitogens, before finally arresting after mitosis. However, more recent reports suggested that the commitment to cycle in response to serum occurs already in G2 phase and requires the Ras-dependent induction of cyclin D1, which promotes following G1/S transition. To test whether this hypothesis applies to arrest induced by contact inhibition, we used an in vitro wounding model where quiescent human dermal fibroblasts, stimulated to proliferate by mechanical injury, synchronously exit cell cycle after mitosis due to renewed confluence. We show that this exit is preceded by p27-dependent inhibition of cyclin A-Cdk1/2, cyclin D1 downregulation and reduced pre-mitotic pRb pocket protein phosphorylation. Over-expression of cyclin D1 but not p27 depletion reversed this phenotype and compromised confluence-driven cell cycle exit. Thus, a balance between cyclin D1 and p27 may provide sensitive responses to variations in proliferative cues operating throughout the cell cycle.     

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1 and Cyclin D1 and induces cell cycle exit and neuronal differentiation

A fundamental question in neurobiology is how the balance between proliferation and differentiation of neuronal precursors is maintained to ensure that the proper number of brain neurons is generated. Substantial evidence implicates DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A) as a candidate gene responsible for altered neuronal development and brain abnormalities in Down syndrome. Recent findings support the hypothesis that DYRK1A is involved in cell cycle control. Nonetheless, how DYRK1A contributes to neuronal cell cycle regulation and thereby affects neurogenesis remains poorly understood. In the present study we have investigated the mechanisms by which DYRK1A affects cell cycle regulation and neuronal differentiation in a human cell model, mouse neurons, and mouse brain. Dependent on its kinase activity and correlated with the dosage of overexpression, DYRK1A blocked proliferation of SH-SY5Y neuroblastoma cells within 24 h and arrested the cells in G₁ phase. Sustained overexpression of DYRK1A induced G₀ cell cycle exit and neuronal differentiation. Furthermore, we provide evidence that DYRK1A modulated protein stability of cell cycle-regulatory proteins. DYRK1A reduced cellular Cyclin D1 levels by phosphorylation on Thr286, which is known to induce proteasomal degradation. In addition, DYRK1A phosphorylated p27^Kip1 on Ser10, resulting in protein stabilization. Inhibition of DYRK1A kinase activity reduced p27^Kip1 Ser10 phosphorylation in cultured hippocampal neurons and in embryonic mouse brain. In aggregate, these results suggest a novel mechanism by which overexpression of DYRK1A may promote premature neuronal differentiation and contribute to altered brain development in Down syndrome.     

p27Kip1 regulates T cell proliferation

Our studies addressed the mechanism by which serum acts in conjunction with T cell receptor (TCR) agonists to promote the proliferation of primary splenic T cells. When added to resting splenocytes, TCR agonists initiated G(0)/G(1) traverse and activated cyclin D3-cdk6 complexes in a serum-independent manner. On the other hand, both TCR agonists and 10% serum were required for the activation of cyclin E-cdk2 and cyclin A-cdk2 complexes and the entry of cells into S phase. Serum facilitated cdk2 activation by maximizing the extent and extending the duration of the TCR-initiated down-regulation of the cdk2 inhibitor, p27(Kip1). Although p27(Kip1) levels were reduced (albeit submaximally) in cells stimulated in serum-deficient medium, nearly all of the cdk2 complexes in these cells contained p27(Kip1). In contrast, in cells receiving TCR agonist and 10% serum, little if any p27(Kip1) was present in cyclin-cdk2 complexes. Unlike wild-type splenocytes, p27(Kip1)-null splenocytes did not require serum for cdk2 activation or S phase entry whereas loss of the related cdk2 inhibitor, p21(Cip1), did not override the serum dependence of these responses. We also found that cdk2 activation was both necessary and sufficient for maximal expression of cdk2 protein. These studies provide a mechanistic basis for the serum dependence of T cell mitogenesis.     

Regulation of p27Kip1 during gentamicin mediated hair cell death

The INK4 and Kip/Cip families of Cyclin Dependent Kinase inhibitors (CKIs) are regulators of the cell cycle. In addition, CKIS including p27^Kip1can protect cells from apoptosis in vitro. However, little is known about protective effect of p27^Kip1in vivo. We used systemic treatment with aminoglycosides to induce hair-cell death in the basilar papilla (BP), the auditory organ of the avian inner ear, and characterised the expression of p27^Kip1with confocal and immunofluorescence microscopy. In contrast to the adult mammalian cochlea where p27^Kip1is expressed only in supporting cells, p27^Kip1is found in the nuclei of both hair cells and supporting cells in the BP of the normal, mature bird. Forty-eight hours after gentamicin treatment, hair cells with TUNEL positive nuclei and hair cells with pyknotic nuclei were both detected, suggesting many hair cells die by apoptosis. When the BP was double labelled for p27^Kip1and myosin VIIa, a hair-cell specific protein, all dying hair cells that had been ejected from the epithelium were found to be myosin VIIa positive but negative for p27^Kip1even though nuclear remnants were still visible. In the transition zone where partial hair-cell loss occurs, freshly ejected hair cells lying immediately above the surface of the BP no longer expressed p27^Kip1. Damaged hair cells within the epithelium in the transition zone contained p27^Kip1in their cytoplasm but not in their nuclei. These data support recent in vitro findings suggesting that p27^Kip1protects cells from apoptosis and that its downregulation may be a general feature of programmed cell death.     

A growth factor-dependent nuclear kinase phosphorylates p27(Kip1) and regulates cell cycle progression

The cyclin-dependent kinase inhibitor, p27(Kip1), which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27(Kip1) is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27(Kip1) and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G(0)/G(1), and expression of hKIS overcomes growth arrest induced by p27(Kip1). Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27(-/-) cells treated with KIS siRNA grow and progress to S/G(2 )similar to control treated cells, implicating p27(Kip1) as the critical target for KIS. Through phosphorylation of p27(Kip1) on S10, hKIS regulates cell cycle progression in response to mitogens.     

p27Kip1 acts downstream of N-cadherin-mediated cell adhesion to promote myogenesis beyond cell cycle regulation

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as "community effect".     

p27Kip1 inhibits the cell cycle through non-canonical G1/S phase-specific gatekeeper mechanism

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 has been shown to regulate cellular proliferation via inhibition of CDK activities. It is now recognized that p27Kip1 can regulate cellular processes through non-canonical, CDK-independent mechanisms. We have developed an inducible p27Kip1 model in cultured cells to explore CDK-independent p27Kip1 regulation of biological processes. We present evidence that p27Kip1 can function in a CDK-independent manner to inhibit entry and/or progression of S phase. Even though this p27Kip1 mechanism is non-canonical it does requires the intact cyclin-binding motif in p27Kip1. We suggest a mechanism similar to that proposed in post-mitotic neural cells whereby p27Kip1 functions to coordinate growth arrest and apoptosis. Our hypothesis supports the concept that p27Kip1 is a gatekeeper for the entry and progression of S phase through interaction with specific protein(s) or via binding to specific DNA sequences in a CDK-independent manner.     

PDK1 regulates cell proliferation and cell cycle progression through control of cyclin D1 and p27Kip1 expression

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).     

p27Kip1 Metabolism: A Fascinating Labyrinth

The progression through the phases of cell division cycle is regulated by different cyclins and cyclin-dependent kinases (CDKs) complexes. Due to their key function, the activity of cyclin/CDK complexes is controlled by several mechanisms, including the inhibition by a number of proteins collectively defined CDK inhibitors or CKIs. Among the CKIs, p27Kip1 represents a protein of central activity for the control of several phenotypes, including proliferation, differentiation and malignant transformation. p27Kip1 belongs to the growing family of “natively unfolded”, “in¬trinsically disordered” or “intrinsically unstructured” proteins. The disorder proteins present a very large number of possible conformations that, after the binding, converge to a well-defined structure with an extraordinary affinity for the target. As matter of fact, the absence of a preexisting folding strongly facilitates p27Kip1 interaction with a number of targets. Until recently, p27Kip1 has been solely viewed as a nuclear protein with the function of modulating cyclin–CDK activity and hence, cell-cycle progression. However, exhaustive studies have now demonstrated that the protein plays additional roles outside of the nucleus, including, particularly, the control of cell motility. Thus, the cellular localization is of fundamental importance in p27Kip1 function. Accordingly, at least two different mechanisms of degradation, occurring either in the nucleus or in the cytosol, have been observed. Convincing evidences have demonstrated that p27Kip1 is a phosphoprotein showing at least 6-8 phosphorylatable residues. However, the precise functional roles of the phosphorylations and the identification of the kinases responsible for the post-synthetic modifications are still debated. In this brief review, we will report the Literature data that connect the post-synthetic modifications of p27Kip1 with its function, localization and metabolism. The picture that emerges demonstrates that several of the pieces of the CKI metabolism are still nebulous.     

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.     

p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells

The cyclin-dependent kinase (Cdk) inhibitors p21(Cip1) and p27(Kip1) have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21(Cip1) and p27(Kip1) in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21(Cip1) or p27(Kip1) arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21(Cip1) arrested cells in both G(1) and G(2) cell cycle phases, p27(Kip1) blocked the G(1)/S-phase transition. Furthermore, although both p21(Cip1) and p27(Kip1) associated with Cdk6, only p27(Kip1) significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21(Cip1) triggered megakaryocytic maturation, whereas p27(Kip1) resulted in the expression of erythroid markers. Consistently, p21(Cip1) and p27(Kip1) were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21(Cip1) and p27(Kip1) in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable.     

Oncostatin M-stimulated apical plasma membrane biogenesis requires p27(Kip1)-regulated cell cycle dynamics

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27(Kip1), which inhibits G(1)-S phase progression. Forced G(1)-S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G(1)-S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27(Kip1)/cdk2-mediated G(1)-S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways.     

Connexin32 inhibits gastric carcinogenesis through cell cycle arrest and altered expression of p21Cip1 and p27Kip1

Gap junctions and their structural proteins, connexins (Cxs), have been implicated in carcinogenesis. To explore the involvement of Cx32 in gastric carcinogenesis, immunochemical analysis of Cx32 and proliferation marker Ki67 using tissue-microarrayed human gastric cancer and normal tissues was performed. In addition, after Cx32 overexpression in the human gastric cancer cell line AGS, cell proliferation, cell cycle analyses, and p21^Cip1 and p27^Kip1 expression levels were examined by bromodeoxyuridine assay,flow cytometry, real-time RT-PCR, and western blotting. Immunohistochemical study noted a strong inverse correlation between Cx32 and Ki67 expression pattern as well as theirlocation. In vitro, overexpression of Cx32 in AGS cells inhibited cell proliferation significantly. G₁ arrest, up-regulation of cell cycle-regulatory proteins p21^Cip1 and p27^Kip1 was also found at both mRNA and protein levels. Taken together, Cx32 plays some roles in gastric cancer development by inhibiting gastric cancer cell proliferation through cell cycle arrest and cell cycle regulatory proteins. [BMB Reports 2013; 46(1): 25-30]     

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.     

CDK inhibitors,p21 and p27, participate in cell cycle exit of mammalian cardiomyocytes

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21^Cip1 and p27^Kip1 but not p57^Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21^Cip1 and p27^Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21^Cip1 and p27^Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21^Cip1 and p27^Kip play important roles in the cell cycle exit of postnatal cardiomyocytes.