PF-3758309, p21-activated kinase 4 inhibitor,suppresses migration and invasion of A549 human lung cancer cells via regulation of CREB,NF-κB,and β-catenin signalings

SUMOylation has been considered as an important mechanism to regulate multiple cellular processes, including inflammation. TAB2 (TAK1-binding protein 2) is an upstream adaptor protein in the IL-1 signaling pathway. Covalent modifications of TAB2 have not been well studied. In this study, we demonstrated that TAB2 could be modified by SUMO. Using Ubc9 (SUMO-conjugating enzyme) fusion and mutation analysis, we identified evolutionarily conserved lysine 329 as the major SUMOylation site of TAB2. PIAS3, a SUMO E3 ligase, preferentially interacted with and promoted its SUMOylation. Interestingly, block of SUMOylation by mutation of lysine 329 enhanced the activity of TAB2, as reflected by AP-1 luciferase reporter assays. Taken together, these results suggest that SUMOylation may serve as a novel mechanism for the regulation of TAB2.     

LIM蛋白KyoT与PcG蛋白的相互作用

KyoT为LIM结构域蛋白 ,可与转录因子RBP J相互作用并抑制RBP J介导的转录激活。为了研究其功能 ,用酵母双杂交法筛选了与KyoT相互作用的蛋白质 ,其中包括同属于PcG (polycombgroup)家族的蛋白质Ring1和hPc2 (humanpolycomb2 )。为进一步验证KyoT2与Ring1和hPc2的相互作用 ,首先在酵母中证实了KyoT2与它们的相互作用并得到阳性结果。然后做GST pulldown实验在体外验证了KyoT2与Ring1和hPc2在体外的相互作用。进而又构建了KyoT2与Ring1和hPc2不同的截短体 ,利用酵母双杂交进一步验证了它们相互作用的区段 ,发现KyoT的LIM结构域与Ring1的全长相互作用 ,与hPc2的C末端和全长相互作用。本研究验证了KyoT2与Ring1和hPc2的相互作用 ,对探讨KyoT的功能以及分析Notch信号途径与PcG家族的关系提供一定的依据。     

Regulation of REGγ cellular distribution and function by SUMO modification

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.     

KyoT3, an isoform of murine FHL1, associates with the transcription factor RBP-J and represses the RBP-J-mediated transactivation

Previously, we have shown that KyoT2, an isoform of the four and a half LIM domain protein 1 (FHL1), modulates Notch signaling via repressing RBP-J-mediated transactivation. In this study, we investigated the effect of another isoform of FHL1, KyoT3, on transactivation of a RBP-J-dependent promoter. We found that KyoT3 was expressed widely in a variety of tissues. By constructing EGFP fusion proteins, we showed that KyoT3 locates preferentially in nucleus. KyoT3 interacted with RBP-J, as shown by co-immunoprecipitation assays. Moreover, we demonstrated by a reporter assay that KyoT3 repressed transactivation of a RBP-J-dependent promoter, which was activated by both the Notch intracellular domain and Epstein-Barr virus nuclear antigen 2, an EB virus-encoded oncoprotein. These results suggest a multi-elemental control of the Notch signaling pathway, which is critical for cell differentiation in development.