April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 128-137, April 2009. ISSN 0973-8916 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 AKTinduced phosphorylation at threonine 145 of p21CIP1 present the function for subcellular localizations in HER2 overexpressing breast 132 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 regulation of CDK inhibitors
Current Trends in Biotechnology and Pharmacy Vol. 3 (2) 128-137, April 2009. ISSN 0973-8916 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 ubiquitindependent 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 G 1 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 0 and falls as cells move through G 1 into S phase. At the same time, the cyclin D/ CDK4 bound p27KIP1 is maximal during early G 1 . 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 downregulating p27KIP1 Phosphorylation affects degradation and localization of p21CIP1 and p27KIP1 proteins Mitogenic signalings often cause downregulation 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 133 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 Jinhwa
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April Journal-2009.p65 - Association of Biotechnology and Pharmacy

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