Regulation of CIP/KIP cell cycle inhibitors and their biological implications
By - 06/28/2009
(1 vote)
Jinhwa Lee
Dept. of Clinical Lab Science, Dongseo University, Busan 617-716, Korea
Running title: regulation of CDK inhibitors
For correspondence:
e-mail address: jinhwa2000@gdsu.dongseo.ac.kr
Current Trends in Biotechnology and Pharmacy , Volume 3 (2) April - 2009
Abstract
The cell cycle regulation is a key homeostatic device upon the cellular decision during the multi processes like proliferation, differentiation, survival and death. Human cancers can arise from the result of functional failure in cell cycle regulators. Therefore, activity of the major cell cycle regulator, cyclin-dependent kinase (CDK), is tightly regulated by cyclin dependent kinase inhibitors (CKIs) such as the p21CIP1 and p27KIP1. These CKIs, mainly functioning as a cyclinE/CDK2 complex inhibitor during the G1 cell cycle, have been reported to play disparate roles including the assembly of cyclinD/CDK4,6 and others that apparently assist cell growth if not help carcinogenesis. While their genetic disruptions are rarely found in human cancers, low expression levels or cytoplasmic mislocalizations of the p21CIP1 and p27KIP1 often correlate with human malignancies. Recent studies show that signalling kinases can directly phosphorylate these proteins as a substrate and change their activities in the role of a cell cycle inhibitor by switching interacting partner proteins after the phosphorylation-driven structural modifications. This report will discuss the complex regulatory mechanisms of p21CIP1 and p27KIP1 proteins on the cue of extracellular signals and their indications of functional importance to carcinogenesis.
Keywords: Cell cycle; Proliferation; CDK, p21CIP1, p27KIP1
Introduction
The mammalian cell cycle operates with four distinct phases, G1, S, G2 and M in a tightly regulated manner, each of which should be completed before the next begins (1). Progression of cell cycle transitions is mediated by sequential activation of the cyclin/CDK complexes. Since timely regulation is absolutely important in normal cell cycle progression, cyclin/CDK complexes are integrators of the multiple signals. Those signals include extracellular signals such as cytokines, hormones, or physical interactions with extracellular matrix or other cells. In mammalian tissues, several cyclin/CDK complexes play a role in the G1-to-S transition (2). In early G1, D-type cyclins are elevated from mitogenic signals and activate CDK4 or CDK6 (3). The three D-type cyclins (D1, D2 and D3) are expressed differentially when CDK4 and CDK6 are co-expressed in many cell types (4). In late G1, cyclin E/CDK2 activity is elevated due to the initial cyclin D/CDK4,6 activation, not by mitogenic stimulation. The increased cyclin E/CDK2 activity phosphorylates pRb and releases E2F transcription factors from an inactive or repressive pRb–E2F complex, which initiates a whole new synthesis of proteins involved in DNA replication (5). The E2F genes also involve some protooncogenes and some cell-cycle regulatory proteins, as well as cyclin E that forms a feedback loop to expedite and commit to enter S phase. Activity of cyclin/CDK complexes is regulated in multiple ways by the accumulation of the cyclin, at the level of assembly into the cyclin/CDK complexes, and by specific phosphorylation and dephosphorylation of the components. One additional and important regulatory mechanism of these G1 cyclin/CDK complexes is their association with CKIs. The CKIs either physically bind and block the cyclin/CDK complexes or inhibit substrate/ATP access. Early investigators postulated that some of these CKIs have additional functions (6). CKIs are grouped to two gene families: the smaller INK4 (inhibitors of CDK4) and the larger CIP/KIP (CDK interacting protein/ kinase inhibitory protein).
The INK4 family is composed of p16INK4A, p15INK4B, p18INK4C, p19INK4D (7). The common structural feature among them is the ankyrin repeats and all of them can inhibit CDK4. They compete with D-type cyclins for binding to the CDK subunit (8). The INK4 family proteins are characterized by specific binding to CDK4 and CDK6. Tissue-specific functions of this family of cell cycle inhibitors have been suggested recently because of the diversity in the pattern of expression of INK4 genes (9). The INK4 proteins are commonly lost or inactivated by mutations in many different cancer types. In addition to their role in arresting cells in the G1-phase of the cell cycle, it also increases interests of researchers that they have been shown to participate in different cellular processes other than cell cycle regulation. The discoveries of involvement of INK4 proteins include their functions in senescence, apoptosis, DNA repair, and multistep oncogenesis. The p16INK4A is a tumor suppressor that inhibits the cyclin D-associated kinase complexes. Loss of function in p16INK4A results in the same effect of loss of function in Rb (10). It has been reported that p14ARF protein is closely related to p16INK4A. The ARF protein, a spliced variant of the INK4A gene, is known to have a function of stabilizing p53 by preventing degradation (11). The second INK4 family protein, p15INK4B, regulates the cell cycle clock by inhibiting cyclinD/CDK4- or cyclinD/CDK6-mediated phosphorylation of Rb. Induction of p15INK4B-triggered G1-phase arrest occurs in response to TGF-β (12). Loss of p15INK4B gene is associated with lymphoproliferative disorders and tumor formation (13). The p15INK4B mediated pathways that control G1/S transition are frequently deregulated in human cancers such as prostate cancer, melanoma, pituitary adenoma, acute myeloid leukemia, gastric cancer. The p18INK4C seems to play an important role in growth control and its expression is found in many different tissue types. It has been suggested that loss of p18INK4C function results in shortening the G1 phase and thus facilitates the cell cycle progression. Interestingly, functional synergism between p18INK4C and p27KIP1 has been implicated in pituitary tumors (14, 15). A recent study of the gene encoding p19INK4D is induced by vitamin D3 derivatives and by retinoids. Therefore, the chemopreventive effects of vitamin D3 may be associated with this induction of p19INK4D expression. Recent studies reported that the knockdown of p19INK4D renders cells sensitive to autophagic cell death (16)
The second group of cell cycle inhibitors is CIP/KIP family proteins: p21CIP1, p27KIP1 and p57KIP2. The structural feature of these proteins shows an ability to bind a whole cyclin/CDK complex, different from INK4 family proteins’ ability to bind CDK protein alone by competing for cyclin’s binding. They can function throughout the all cell cycle phases by interacting with different kinds of cyclin D, E, A/CDK complexes. Although initially identified as cell cycle inhibitors, these CIP/KIP family proteins have emerged to display roles in different cellular functions ranging from apoptosis to cell migration and have appeared to add their lists on other important cellular functions. These functions are essential for the maintenance of normal cell homeostasis. The first identified inhibitor in this family is p21CIP1 that is also named SDI1 (senescent cell-derived inhibitor 1) or WAF1 (wild-type p53-activated fragment 1). The existence of p27KIP1 is discovered by TGF-b induced G1 arrest and its function as a G1 arrest controller diversifies into cases such as contact inhibition mediated G1 arrest. p57KIP2 that is discovered during the search for homologues of p21CIP1 and p27KIP1 also participates in the control of cell cycle regulation as well as differentiation and apoptosis in particular tissues (17). Recent studies demonstrate that p57KIP2 functions in many different cellular processes beyond cell cycle control. In Schwann cells, the myelinating glial cells of the peripheral nervous system, small hairpin RNA dependent suppression of p57KIP2 results in cell cycle exit and the initiation of the cellular differentiation program via p57KIP2/LIMK-1 interactions (18). While function and regulation of p57KIP2 are under active investigations, this manuscript will focus on the p21CIP1 and p27KIP1 proteins that have recently been demonstrated extensively for their regulatory mechanisms with relation to cellular transformation into cancer.
Upon the exposure of cells to growth inhibitory signals, p21CIP1 and p27KIP1 bind to cyclin/CDK complexes to inhibit cyclin/CDK catalytic activity and result in cell cycle arrest. It becomes evident that p21CIP1and p27KIP1 might have new activities that are unrelated to their function as CDK inhibitors. From the help of copious publications, the importance of cytoplasmic localization and the identification of new targets have revealed novel functions for these p21CIP1 and p27KIP1 proteins beyond cell cycle controls. A complex signaling and phosphorylation network modifies these proteins and changes their degradation, subcellular localization, and protein-protein interactions. This article will focus on reviewing the cellular functions and recent advances of the p21CIP1 and p27KIP1 proteins.
A CIP/KIP protein interacts with cyclin/CDK complexes
G1 cell cycle progression relies on the sequential activation of G1 cyclin/CDK complexes to enter the S phase. Tight regulation of G1 cyclin/CDK complexes therefore is essential when cells decide to divide because the commitment site, restriction point, for cell division lies at late G1. INK4 family proteins bind and inhibit CDK4 and CDK6 specifically and CIP/KIP proteins interact with the cyclin E/CDK2 complex, inhibiting cell cycle transition from G1 to the S phase (19). Different from the INK4 family proteins, CIP/KIP proteins do not dissociate cyclin/CDK complexes (20). The first α-helical loop of a CIP/KIP protein interacts with the cyclin, and the second helix binds to the catalytic cleft of the CDK subunit, thereby blocking ATP loading (21, 22). Many cyclinD/CDK4,6 complexes have been found to contain p21CIP1or p27KIP1 and surprisingly maintain the active state of the cyclin D/CDK4,6 complex. With the help of data from animals of the knockout p21CIP1 and p27KIP1 genes, p21CIP1and p27KIP1 proteins are now believed to facilitate assembly of the two subunits of cyclin D1 and the CDK4 or CDK6 (5). Then these contradictory dual functions of p21CIP1 and p27KIP1 comprise inhibition of the nuclear CDK2 and assembly, thus activation, of cytosolic CDK4 or CDK6 with cyclin D. Therefore, the p21CIP1 and p27KIP1 proteins might have totally irrelevant functions in different intracellular locations and their structural modifications or interacting proteins might also be differentiated according to the localization.
In addition to the assembly, cytoplasmic CIP/KIP proteins also promote the nuclear accumulation of D-type cyclins. p27KIP1 protein can bind to the nuclear pore-associated protein mNPAP60, and interact with the nuclear export protein CRM1 (23, 24). p21CIP1 blocks the interaction between cyclin D1 and the CRM1, leading to increased cyclin D levels in the nucleus. Since nuclear export is mediated by CRM1, CRM1 interaction with p21CIP1 and p27KIP1 causes trans-localization into the cytoplasm. It can also be interpreted that the interaction displaces cyclin D1 from CRM1 to increase the cyclin level in the nucleus. The binding ability of the CIP/KIP proteins to cyclin D/CDK4,6 complexes can be viewed cyclin D/CDK4,6 complexes as a sequestering station for the CIP/KIP cell cycle inhibitors, thus freeing and activating the cyclin E/CDK2 complex. p16INK4A null mouse shows a high incident of tumor development and p16INK4 null phenotype is similar to the null phenotype of pRb. The INK4-free cyclin D/CDK4 complexes can sequester the CIP/KIP proteins, freeing cyclinE/CDK2 and resulting in hyperphosphorylation of Rb from the inhibitor-free cyclinE/CDK2 activity. Different from the p16INK4 null mouse phenotype, p27KIP1 null mice do not show an increased tumor frequency. However, null mice of multiple cell cycle inhibitors display severe developmental defects although null phenotypes show clear discrepancies between INK and CIP/KIP family proteins, suggesting the existence of redundant roles.
The CIP/KIP family proteins are best known as cell cycle inhibitors but they also play a role in cell differentiation, senescence and apoptosis. p21CIP1 is involved in p53-dependent DNA damage-induced G1 arrest. The main role of p21CIP1 of course is G1 arrest through inhibiting the activity of cyclinE/CDK2. Cytokines such as TGF-beta, TNF, or IL6 induce p53-independent expression of p21CIP1 which can suppress apoptosis and at the same time cell cycle inhibition (25). Since p21CIP1 functions in a variety of different cellular processes, the consequences of changes in p21CIP1 regulation after DNA damage are complex. Previous reports indicate that p21CIP1 plays both anti- and proapoptotic roles. Cytoplasmic p21CIP1 can interact with ASK1 and procaspase 3 to suppress apoptosis. On the other hand, overexpression of p21CIP1 and retinoic acid-induced p21CIP1 promote apoptosis. In many cell types, p27KIP1 plays a key role in the decision to the G1-S entry. First identified as a CDK2-inhibitor in contact inhibition or TGF-beta arrested cells, 27KIP1 is also induced by other anti-mitogenic signals such as cAMP and rapamycin or lovastatin treatment. Decreased expression of p27KIP1 in breast cancer cells correlates with poor prognosis.
CIP/KIP protein phosphorylation by mitogenic, antimitogenic and other signaling pathways
CIP/KIP proteins are able to respond to diverse extracellular demands and help cells become fit to the new environment through proper cell cycle regulation. On performing the critical actions, p21CIP1 and p27KIP1 proteins share common cell cycle effects as they have conserved sequences in their inhibitory domains and both proteins can form a ternary complex with cyclin/CDK in response to extracellular signals. Both p21CIP1 and p27KIP1 proteins are short-lived: activity of these proteins largely depends on the protein levels that are regulated mainly through proteasome-dependent degradation and/or transcriptional control. Still under active uncovering of mechanisms involved, these proteins possess seemingly contradictory actions of facilitating cell motility and interacting with proteins involved in functions aside from cell cycle regulations when they are localized in the cytoplasm. Fibroblasts are the model system that is mainly used to study the activities of p21CIP1 and p27KIP1 and their role on cell cycle control. In other cell types, p21CIP1 and p27KIP1 are unusually controlled by different regulatory curcuits that are sometimes contrasting in functions between these two proteins. As p21CIP1 and p27KIP1 knock-out mice display different phenotypes, it is not unusual that p21CIP1 and p27KIP1 demonstrate nonoverlapping and distinctive functions (26). Significance of multiple biological functions such as apoptosis and differentiation of these two p21CIP1 and p27KIP1 proteins are gradually acknowledged. The onset of environmental cues delivers these cell cycle inhibitors to a new biological role following posttranslational modifications like phosphorylation or ubiquitination (27). Extensive studies have informed us that p21CIP1 and p27KIP1 proteins remodel themselves predominantly through phosphorylation and consequent alteration of their interacting protein partners, resulting in the changes in cellular functions and localization as well as their protein levels.
It is well known that induced phosphorylation by growth factors activates cytoplasmic protein kinases such as Raf, MEK, ERK, JNK, p38 MAPK or SAPK, JAK, AKT and other kinases. Nonetheless, it is interesting to know that some of them directly phosphorylate CKI as exampled above in the case of p21CIP1 and p27KIP1 by using the cell cycle inhibitors as their own substrates. It is more important to know the condition, function and the precise phosphorylation sites of the substrate in order to understand how individual signaling network can communicate with these cell cycle regulators. Protein kinases which interact and phosphorylate directly p21CIP1 and p27KIP1 proteins have been studied intensively during recent years using in vivo or in vitro studies (28). Glycogen Synthase Kinase 3 beta phosphorylates p21CIP1 and enhances proteasomal degradation after UV irradiation. PIM-1 Kinase phosphorylation of p21CIP1 promotes stability of p21CIP1 in H1299 cells. AKT phosphorylation of p21CIP1 functions in increasing protein stability of p21CIP1 and cell survival (29). Other studies regarding AKT-induced phosphorylation at threonine 145 of p21CIP1 present the function for subcellular localizations in HER2 overexpressing breast cancer cells. Protein phosphorylation has an essential function in all kinds of cells but the delicate regulation pattern tells us that the same kinase-substrate interaction does not always aim for the same functional outcome; these examples are found more frequently when other cell types or different conditions even with the same cell types are used. When a kinase phosphorylates the substrate on multiple sites, one site can be more phosphorylated than others in a certain condition probably because of the presence of the third protein or small molecules that affect interaction between the kinase-substrate. Phosphorylation and functional changes like cytoplasmic localization of p21CIP1 with HER2 overexpression in breast cancer cells impose clinical values (30). The very kinase critical for transformation therefore becomes an important question to answer; however, there might be more than one kinase for the final target of CIP/KIP proteins because both p21CIP1 and p27KIP1 proteins possess multiple phosphorylation sites. Cytokines inducing differentiation also end up on phosphorylating the same targets as antimitogen signals. Myoblast cell survival has been reported to be promoted by p21CIP1 protein through the MAPK cascade activation (31). It would be biologically meaningful to discover the phosphorylation sites and the kinases for the p21CIP1 protein during this myocyte differentiation. In K562 human leukemia cells, both p21CIP1 and p27KIP1 are involved in the regulation of cell cycle progression and differentiation. Interestingly, there is a difference in the biological effects as p21CIP1 directs cells toward megakaryocytic differentiation and p27KIP1 provokes an erythroid differentiation response.
Important roles of p27KIP1 on guarding cells against breast cancer have advanced our understanding in relationship between the phosphorylation and regulation of the inhibitor protein. Phosphorylation on serine/threonine of p27KIP1 by ERK1 is a signal for ubiquitination and phosphorylation on more than one threonine sites by different kinases involves cytoplasmic localization. CyclinE/CDK2 phosphorylates p27KIP1 on Thr187 and leads to ubiquitin-dependent phosphorylation. p27KIP1 protein phosphorylated by AKT at Thr157 and Thr198 becomes better assembler of cyclin D/CDK4 complex formation (32, 33). Since p27KIP1 binding to cyclin D/CDK4 facilitates activation of cyclinE/CDK2 through sequestration of the inhibitory protein, the differential binding of p27KIP1 to the distinct CDKs during G1 cell cycle can be attributed to the phosphorylation status of p27KIP1. Altered p27KIP1 phosphorylation would then switch p27KIP1 cyclin/complexes. As p27KIP1 phosphorylation is cell cycle dependent, the cyclinE/CDK2 inhibitory activity of p27KIP1 is maximal in G and falls as cells move through G1 into S phase. At the same time, the cyclin D/CDK4 bound p27KIP1 is maximal during early G1. Anti-mitogenic signaling dissociates p27KIP1 from CDK4/6 complexes and accumulates in cyclinE/CDK2. As with Thr145 phosphorylation of p21CIP1 by PIM1, PIM kinases promote cell cycle progression by phosphorylating and down-regulating p27KIP1
Phosphorylation affects degradation and localization of p21CIP1 and p27KIP1 proteins
Mitogenic signalings often cause down- regulation through accelerated proteolysis and mislocalization out of nucleus into cytoplasm of p21CIP1 and p27KIP1 proteins. The fact that mutation or deletion of p21CIP1 and p27KIP1 genes is uncommon in human cancers suggests that post-transcriptional regulatory mechanisms may be more important in the process of cancer development. These proteins are short lived and their expression is tightly regulated by proteasome-mediated protein degradation. The ubiquitination-dependent degradation pathway involves E3-ubiquitin ligases, such as SCFSKP2 (34). While less often than accelerated proteolysis in human cancers, cytoplasmic localization of p27KIP1 – that of p21CIP1 in cancers is less understood- has been observed in some advanced cancers. Many tumor suppressor proteins including p53, FOXO family gene products, p21CIP1 and p27KIP1 proteins function when they are present in the cell to prevent cancer initiation or progression. Inhibition of FOXO family proteins that compose p21CIP1 and p27KIP1 gene transcription factors can result in the negative transcriptional regulation of p21CIP1 and p27KIP1 proteins. Therefore, mislocalization of those nuclear proteins including FOXO family proteins into the cytoplasm can disable them as a tumor suppressor. In the cytoplasm, FOXO family proteins can no longer exercise a transcription factor, nor do the p21CIP1 and p27KIP1 proteins the cyclinE/CDK2 inhibitor. Export of nuclear proteins generally involves modification in the leucine-rich nuclear export signal sequences which allows binding to the CRM1/RanGTP proteins and then the nuclear proteins are ready for the journey out of nucleus to the cytoplasm. SCFSKP2 and CRM1 proteins are notably recognized in the study of cancer development for this very reason that they help nuclear tumor suppressive proteins evacuate from the functional site, nucleus.
The p21CIP1 protein level is mainly controlled at the transcriptional level. Nonetheless, the fact that a half-life of p21CIP1 is less than 30 min dictates p21CIP1 stability as a considerable control site. Cytoplasmic localization of this nuclear CDK inhibitor of the p21CIP1 protein can be added as another regulatory site. Modification of p21CIP1 by phosphorylation that changes interaction with other cellular proteins is one mechanism to control the protein level in general by qualifying the protein for ubiquitin-dependent proteosomal degradation and by mislocalization into cytoplasm. Degradation of p21CIP1 protein involves ubiquitination-dependent and -independent proteasomal targeting. This occurs through binding of its COOH terminus with the C8 subunit of the 20S core of the proteasome. Mitogenic signaling induced phosphorylation of p21CIP1 is precedent in this process. CDK2, AKT, p38MAPK or SAPK, JNK, GSK-3ß, PKA, and PKC have been shown to phosphorylate p21CIP1. PKC can phosphorylate Ser146 in the COOH terminus of p21CIP1 and facilitate degradation. Phosphorylation of p21CIP1 affects interaction with its binding partners, regulating the stability of the protein and its subcellular localization. AKT phosphorylates p21CIP1 at Thr145 and Ser146 within the binding site of PCNA which is known to promote the ubiquitination and degradation of p21CIP1. Phosphorylation at Ser146 by AKT significantly increases the p21CIP1 protein level and Thr145 phosphorylation by AKT prevents PCNA binding and promotes the nuclear export of p21. A link between intracellular localization and proteolysis is better identified for the p27KIP1. Antimitogenic signaling induced phosphorylation of p21CIP1, as predicted, inhibits p21CIP1 degradation. p38MAPK or SAPK and JNK1 activated by TGFß -1 phosphorylates p21CIP1 at Ser130 and increases p21CIP1 stability.
Activity of the target protein CDK2 can affect p21CIP1 stability. While it is intriguing to understand how CDK inhibitors can put the CDK activity to use for their degradation, a novel idea has been recently reported with an introduction of a model of high affinity binding motif, cy1, and low affinity binding motif, cy2, for CDK on p21CIP1 (35, 36). In the model, CDK2 may promote p21CIP1 degradation in a sequential pathway. CDK2-dependent phosphorylation of p21CIP1 at Ser130 would be recognized by a SKP2-containing SCF complex, and ubiquitinated and degraded by the proteasome. Direct phosphorylation on the Thr145 of p21CIP1 by PIM-1 stabilizes it and results in a shift in the subcellular localization of p21CIP1 in H1299 cells (29). The finding that p21CIP1 phosphorylation at Ser114 by GSK-3ß is critical for p21CIP1 protein degradation by UV shows an example of proteosomal degradation of p21CIP1 protein without ubiquitination.
While clinical importance of a cytoplasmic mislocalization of p21CIP1 in tumors is not understood as well as that of p27KIP1, p21CIP1 protein might be more involved in apoptosis compared to p27KIP1 protein. For p27KIP1, cytoplasmic localization is closely linked to poor prognosis in human breast, colon, ovarian, thyroid and esophageal cancers and low protein levels have been identified and associated with tumor progression and poor prognosis in many different cancers including colon, breast, prostate, ovarian, and brain cancer. At G, p27KIP1 stabilization is a result of Ser10 and Thr198. Ser10 phosphorylation in quiescent cells is attributed to MIRK/DYRK1B. Ser10 phosphorylation interferes with the binding of p27KIP1 to cyclin/CDKcomplexes which may reduce p27KIP1 stability. At mitogenic signal, KIS is responsible for Ser10 phosphorylation and becomes an important regulator of p27KIP1 (37). Phosphorylation at Ser10 triggers the export of p27KIP1 from the nucleus into the cytoplasm by a CRM1-mediated export pathway. Therefore, Ser10 phosphorylation results in nuclear export on the mitogenic cues which might expose the protein to the cytoplasmic proteosome and therefore indirectly decrease the stability of p27KIP1 (38). Thr157 located within the nuclear localization sequence of p27KIP1 can be phosphorylated by AKT. It is found that phosphorylation at Thr157 and Thr198 cooperates to enhance cytoplasmic localization of p27KIP1. In addition to the threonine/serine phosphorylation sites, p27KIP1 possesses three tyrosine residues, Tyr74, Tyr78, and Tyr79, all of which are phosphorylated by SRC family kinases. These phosphorylation sites are in the CDK binding region of p27KIP1 and the phosphorylation of these tyrosines makes p27KIP1 unstable. The protein unstability is because p27KIP1 is a poor inhibitor of CDK2 and thus partially restore the kinase activity after tyrosine phosphorylation. Phosphorylation at Thr187 of p27KIP1 by cyclinE /CDK2 complexes provides a binding site for the SCFSKP2 E3 ubiquitin ligase (39). Therefore, p27KIP1 protein levels significantly decrease when cyclinE/CDK2 is activated.
Conclusion
Understanding the p21CIP1 and p27KIP1 protein regulation provides a better insight on the cell cycle regulation mechanisms in human cancer. As their primary function as a CKI is to bind and inhibit a cyclin/CDK complex, these two p21CIP1 and p27KIP1 proteins function throughout the all cell cycle phases by interacting with different kinds of cyclin D, E, A/CDK complexes. These cell cycle inhibitors have emerged to display roles in other cellular functions such as apoptosis and cell migration. Their functions are differentiated through exchanging partner proteins. Therefore a competent structure of these proteins for punctual or scrupulous partner proteins may be critical for the maintenance of normal cell homeostasis. After de novo synthesis, p21CIP1 and p27KIP1 proteins are extensively modified by post-translational modifications of phosphorylation. Phosphorylation of these proteins has been recently reported to be the result of direct substrate-kinase interactions of major signalling molecules such as MAPK or AKT as a final molecular event of extracellular signaling pathways. Mitogen- or antimitogen-induced phosphorylation causes alteration in expression levels and the intracellular location of p21CIP1 and p27KIP1 proteins. The mechanisms involved in cytoplasmic localization as well as degradation of these proteins are important in understanding many human carcinogenesis.
Acknowledgement
This work was supported by grants from the Korea Science and Engineering Foundation (No. R13-2002-020-02001-0, 2007).
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