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Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle whose activities are controlled by associated cyclins. PFTK1 shares limited homology to CDKs, but its ability to associate with any cyclins and its biological functions remain largely unknown. Here, we report the functional characterization of human PFTK1 as a CDK. PFTK1 specifically interacted with cyclin D3 (CCND3) and formed a ternary complex with the cell cycle inhibitor p21 Cip1 in mammalian cells. We demonstrated that the kinase activity of PFTK1 depended on CCND3 and was negatively regulated by p21 Cip1 . Moreover, we identified the tumor suppressor Rb as a potential downstream substrate for the PFTK1/CCND3 complex. Importantly, knocking down PFTK1 expression by using siRNA caused cell cycle arrest at G1, whereas ectopic expression of PFTK1 promoted cell proliferation. Taken together, our data strongly suggest that PFTK1 acts as a CDK that regulates cell cycle progression and cell proliferation.cyclin D3 ͉ p21 ͉ retinoblastoma ͉ cell cycle

Post-translational modification by ubiquitin (Ub) and Ub-like modifiers is one of the most important mechanisms regulating a wide range of cellular processes in eukaryotes. Through mediating 26S proteasome-dependent degradation of substrates, the covalent modification of proteins by multiple Ub (ubiquitination) can regulate many different cellular functions such as transcription, antigen processing, signal transduction and cell cycle. To better understand ubiquitination and its functions, proteomic approaches have been developed to purify and identify more protein substrates. The S5a subunit of the 26S proteasome binds to poly-Ub chains containing four or more Ub. In this study, immobilized GST-S5a fusion protein was used to affinity-purify ubiquitinated proteins from Chang liver cells. The purified proteins were then identified with multi-dimensional LC combined with MS/MS. Eighty-three potential ubiquitination substrates were identified. From these proteins, 19 potential ubiquitination sites on 17 potential substrates were determined. These potential ubiquitination substrates are mainly related to important cellular functions including metabolism, translation and transcription. Our results provide helpful information for further understanding of the relationship between ubiquitination machinery and different cell functions.

The yeast Ga subunit, Gpalp, plays a negative role in the pheromone response pathway. The gpal Val50 mutant was previously shown to have a growth defect, consistent with the GTPase defect predicted for this mutation, and greatly reduced mating. Various explanations for the mating defect have been proposed. One approach to analyze the gpal Val50 mating defect involved epistasis analysis. The low mating of the gpalVal5o mutant was independent of the pheromone receptor; therefore, it results from intracellular activation of the pathway, consistent with a GTPase defect. This result suggests that gpalVal50 mating occurs through the default rather than the chemotropic pathway involved in pheromone response. We therefore tested the effect of a spa2 mutation on gpalVal50 mating, because Spa2p has been implicated in the default pathway. The spa2 mutation greatly reduced the mating of the gpalVal5o mutant, suggesting that gpalVal5o mating occurs predominantly through the default pathway. In a second approach to investigate the gpa[Val5O phenotypes, suppressors of the gpalVal50 mating defect were isolated. Two suppressor genes corresponded to SONI / UFD5 and SEN3, which are implicated in ubiquitin-mediated proteolysis. On the basis of these results, we suggest that a positive component of the default mating pathway is subject to ubiquitin-mediated degradation.