Cell Cycle Progression In G2/M Phase Induced By PKC-MPF In NIH3T3 Cells

The term protein kinase C ( PKC ) stands for a group of at least twelve serine /threconine kinases that are characterized by a high degree of similarity in their catalytic kinase domains and cysteine - rich regions. The PKC family is subdivided into three groups; the classical PKC members (abr) are Ca2 + and diacylg-lycerol - dependent, the novel PKCs are Ca2+ -independent but diacyl-glycerol -dependent and the atypical PKCs are not activated by Ca2+and diacylglycerol in vitro . This may account for the multiplicity and diversity of the cellular activities implicted to be mediated by PKC, since individual PKC enzymes are thought to execute distince cellular functions, at different cellular locations. Harald Mischak ,et al found that in NIH3T3 cells express high amounts of PKC -m RNA ,low but detectable amounts of PKC - 8and PCKe ,and barely detectable amounts of PKC-and-m RNA. PKC-rand - ?m RNA could not be detected at all. Using Western blot analysis , they found that PKC -a was the only member of the PKC family that was present in abundant a-mounts. Low amounts of PKC - s and sometimes PKC - 8 and - could be detected , while antibodies against all other isozymes did not detect PKC - specific bands.According to the current model of cell cycle control, transition between different cell cycle phases is regulated at checkpoints. There are two key checkpoints ;G1/S and G2/M. Progression through the cell cycle is mediated by the activation of a highly conserved family of protein kinases, the cyclin - dependent kinases ( cdks). Activation of a cdk requires binding to a specific regulatory subunit , termed a cyclin. Together, these cyclin/cdk complexes are the universal cell cycle regulators ,with each complex controlling a specific transition between the subsequent phases in the cell cycle . Now a number of different cyclin/cdk complexes have been identified and purified. CHIYA KOSAKA et al reported that phorbol 12 - myristate 13-acetate ( PMA) and 1,2- dioctanoyl - sn - glycerol( DiC8) ,a membrane - permeable DAG,block the G1/S transition by inhibiting the expression of cyclin A and the phosphorlation of the retinoblastoma gene product (pRb) in human vascular endothelial cells and suggested that the inhibition of the activity of cdk2,a cyclin -dependent kinase (cdk) asssociated with cyclin A, is the cause of the PMA - induced Gl/S arrest. In addition , cdk4 and cdk6 bind to cyclin D also regulate Gl phase.The transition from G2/M phase is also mediated by cdk(s). A critical and central component is likely to be MPF ( M - phase promoting factor, another name is maturation - promoting factor, it was first discovered by Masui and Marikert in 1971 ,and then purified from Xenopus laevis eggs in 1988. MPF is a complex that consists of the protein kinase p34cdc2 and the regulatory protein cycli B; the protein kinase activity of p34cdc2 is strictly dependent on its association with cyclinB. In Xenopus laevis eggs the absent of cyclin B, MPF will not be activited. The amounts of cyclin B changes due to the phase of cell cycle. In Gl phase ,cyclin B start to be synthesized, getting more and more and the highest of amounts in the late of G2 phase . the amounts of cyclin B is the necessery for the activity of MPF. In addition to cyclin B, p34cdc2 can be activity or in activity in three site Thrl4,Tyrl5 and Thr 161. In all eukaryotic cells that have been studied , protein phosphorylation serves as a key regulator of cell cycle events . Entry and exit from the M phase of the cell cycle are triggered universally by the activation and inactivation of p34cdc2, a cyclin - dependent kinase that binds B -type mitotic cyclins. Only when Thrl4 and Tyrl5 are in dephosphorylation and Thrl61 is in the state of phosphorylation ,p34cdc2 is activity.In addition, activation and inactivation of p34cdc2are induced by Ca2+and prevented by Ca2+ chelators in permeabilized cells and cell - free systems. This suggests that intracellular Ca2+ transients may play an important physiological role in the control of p34cdc2 kinase activity .