The Interaction Between ARPC2 And PRAK

As key components of signal transduction systems in organisms, mitogen-activated protein kinases (MAPKs) are considered to be involved in many cellular processes and pathogenesis of many severe diseases. p38, a subfamily of MAPKs, is involved in the stress and growth factors-induced gene expression and the regulation of cell proliferation and cell apoptosis, as well as in the regulation of cytoskeleton remodeling. p38 can activate MAPKAP-K2/3 and PRAK, which in turn activate HSP27, leading to cytoskeleton remodeling. Although it is widely known that PRAK is involved in cytoskeleton remodeling, the exact mechanism is little elucidated. To investigate the function of PRAK in vivo, a GST fusion protein of PRAK was used as a bait and screened through T7 phage display system. It was found that PRAK may interact with ARPC2 ?a subunit of Arp2/3 complex, which is an important protein complex in the actin cytoskeleton assembly. Then, in order to validate the interaction between these two proteins, in vitro binding and in vivo binding were executed, and the influential factors of the binding were investigated.In the T7 phage display screening in which PRAK as the bait, 13 sequences of coded protein were found, and ARPC2 was one of them. Then, human lung cDNA library was constructed and ARPC2 gene was amplified by specific primers from the library, and was cloned into pET-14b and pcDNA3 vectors, respectively.In the in vitro binding experiment, GST-PRAK and His-ARPC2 were expressed in E. coli strain BL21 and DE3, then purified with GST seprose and Ni-NTA resin, respetively. When these two purified protein were accessible, the in vitro binding was performed. The results showed that GST-PRAK could be co-precipitated by His-ARPC2, which pre-bound to the Ni-NTA resin, and the GST protein alone could not in the same condition, which suggested that ARPC2 could bind to PRAK in vitro.In the in vivo binding experiment, HA-PRAK and FLAG-ARPC2plasmids were co-transfected into NIH/3T3 cells, then the cells were lysed and the supernatant was used in co-immunoprecipitation. The co-immunoprecipitated results through anti-FLAG antibody beads showed that ARPC2 and PRAK could not bind to each other without stimulation or with the stimulation of insulin, but could bind to each other with the stimulation of arsenite. The co-immunoprecipitated results through anti-HA antibody magnetic beads showed the same results. Because of the ability of binding to Arp2/3 complex, GST-WA fusion protein was used to immunoprecipitate endogenous ARPC2, and co-immunoprecipitated endogenous PRAK was detected, which further confirmed that interaction did exist between ARPC2 and PRAK. It was also the most direct evidence of these two proteins interacted with each other. After NIH/3T3 cells were stimulated with different time, the time course of the binding between ARPC2 and PRAK was studied. The results showed that the binding intensity of these two proteins gradually increased upon stimulation, and reached the peak when stimulated 60~90 min approximately, then descended gradually. Whether different stress stimuli could also improve the binding between ARPC2 and PRAK was investigated, and the results indicated that different stress could lead to interaction between these two proteins in a variant degree, and the effect of arsenite, sorbitol and H2C2 were relatively strong among them.The influential issues on the binding between ARPC2 and PRAK were then studied. Firstly, NIH/3T3 cells were pretreated with SB203580, the specific inhibitor of p38 pathway, and PD98059, the specific inhibitor of ERK pathway, and it is shown that these two inhibitors had no effect on the binding. Then, PRAK(182A), the dominant negative mutant of PRAK, PRAK(182D), the dominant positive mutant of PRAK, and PRAK(KM), the loss-of-activity mutant of PRAK, were co-transfected with FLAG-ARPC2 respectively. The responsive pattern of PRAK(182A) was similar to that of wild type PRAK. But to our surprise, PRAK(182A) and PRAK(KM) were found to bind to ARPC2 intensively, but the bindings wer...