Identification Of Interacting Proteins Of PRAK By TAP

p38 regulated/activated protein kinase (PRAK) was originally described as a protein kinase activated downstream of the p38 mitogen-activated protein kinase (p38 MAP kinase/p38 MAPK). PRAK is a member of MAPK-activated protein kinase (MAPKAP kinase/MK) family. A PRAK gene does not appear to be present in either C.elegans or Drosophila, but orthologus are found in most vertebrates. The molecular weight of PRAK is about 54 kD. The human PRAK gene encodes two differentially spliced transcripts giving rise to proteins of 471 amino acids and 473 amino acids, respectively. The two isoforms differ such that only two additional amino acids are found within the C-terminal extension of PRAK of transcript variant 2 when compared to transcript variant 1. The functional significance of these different splice variants is as yet unknown. PRAK was found to be expressed in all human tissues and cell lines examined, with predominant expression in the heart and skeletal muscle. When over-expressed, PRAK localizes to the nucleus of quiescent cells, but upon cellular stress, PRAK translocates to the cytoplasm. While the subcellular localization of endogenous PRAK is somewhat controversial with reports that the endogenous protein is either nuclear or cytoplamic. The activation mechanisms, substrates, and functions of PRAK are still poorly understood.Early data has shown that with the stimulation of cellular stress or proinflammatory cytokines, PRAK was phosphorylated on Thrl82 by p38 MAPK, then activated PRAK phosphorylates small heat shock protein HSP27, resulting in cytoskeletal reorganization and responses to cellular stress. p38 MAPK-mediated phosphorylation of PRAK can lead to redistribution of a significant fraction of PRAK from the nucleus to the cytosol. But it remains unclear what is the function of PRAK translocation. Despite the fact that when over-expressed in cells PRAK can clearly be activated by p38 MAPK, doubts began to surface as to whether or not PRAK is a target of the p38 MAPK pathway in vivo. Recent data indicated that no significant activation of endogenous PRAK could be detected in response to either cellular stress or inflammatory cytokines, despite the concomitant activation of p38 MAPK, and endogenous PRAK was not able to phosphorylate HSP27 in vitro and in vivo. Furthermore, an analysis of protein-protein interactions between endogenous PRAK and p38 MAPK failed to find any evidence for complex formation. Therefore, it is necessary to reevaluate the relationships between p38 MAPK and PRAK, and the substrates of PRAK. Recently, some investigators proposed that phosphorylation of p53 by PRAK following activation of p38 MAPK by Ras plays an important role in Ras-induced senescence and tumor suppression.Other regulators of PRAK may be atypical mitogen-activated protein kinase (atypical MAPK) ERK3 and ERK4. It had been observed that PRAK was uniquely regulated and activated following complex formation with the atypical MAP kinases ERK3 or ERK4. In addition, some experiment showed that knockout of PRAK in mice would result in embryonic lethality. Interestingly, the time of embryonic death corresponds well the peak of ERK3 mRNA expression during mouse embryonic development. Therefore, it was speculated that PRAK participated in ERK3/4-mediated regulation of embryonic development. But it is still unclear what is the substrates of PRAK in performing the functions.In summary, PRAK may exert comprehensive and complex biological functions. But so far our knowledge about PRAK is still very limited, and there are still a lot of controversies about PRAK. In addition, whether there are some unknown regulation mechanisms and functions of PRAK remains to be studied.A protein complex is a functional unit of proteins. In many cases, the subcellular distribution, translocation and protein-protein interactions are the key mechanisms for realization of proteion’s functions. Therefore, the studies on interactions between PRAK and its interacting partner proteins will certainly help understanding of PRAK’s biological functions and their molecular mechanisms. Employment of tandem affinity purification (TAP) combined with mass spectrometry (MS) is a new strategy for purification and identification of protein complexes. The key feature of this technique is the use of two different affinity purification tags that are fused to at least one known component of the protein complex of interest by genetic methods. Performing two consecutive purification steps using affinity purification tags that have gentle washing and elution conditions allows for isolation without disrupting the targeted complex. The main advantage of TAP system is the offer of information about natural protein-protein interactions at physiological states.In this study, we employed InterPlay Mammalian TAP System created newly by Stratagene company to characterize PRAK interacting partners. The streptavidin binding peptide (SBP) coupled with the calmodulin binding peptide (CBP) module of InterPlay Mammalian TAP System does not require a tobacco etch virus (TEV) protease cleavage step to release the complex after the first level of purification, which makes the purification protocol of protein complexes simpler and more economical, and prevents MS analysis from interference resulting from protease pollution. In addition, SBP has less damage to folding of the bait protein and its activity, which is beneficial from its smaller molecular tag.In this study, firstly, human PRAK coding region was subcloned into vector pNTAP to construct a recombinant plasmid pNTAP-PRAK, then Western blot and immunofluorescence assay were used for observation of the expression and subcellular distribution of fusion protein TAP-PRAK. The results showed that the fusion protein could be expressed efficiently, TAP tags had no interference on localization of PRAK, and fusion protein didn’t change the antigenicity of CBP tag.Secondly, HEK293-TAP-PRAK cell line expressing fusion protein stably and at a low level was successful established by G418 screening following a transfection of the recombinant plasmid pNTAP-PRAK into HEK293 cells, to avoid non-natural or non-specific protein-protein interactions resulting from over-expression of bait protein in a transient transfection. To avoid the false-positive results caused by non-specific combinations of TAP tags with irrelevant proteins as much as possible, we established a HEK293-TAP cell line only expressing TAP tags as the negative control system for TAP.Then, we used the positive control vectors of TAP system to evaluate the purification efficiency of TAP, and the results of the preliminary experiment showed that our performance on TAP was satisfactory.At last, we used TAP system followed by DIGE and MS to identify differential protein partners of PRAK in HEK293-TAP-PRAK cell line under NaAsO2 stimulation and non-NaAsO2 stimulation states. In this study, we got 12 candidate interacting proteins of PRAK. These proteins included transcription factors/zinc finger protein, protein kinase, proteins involved in the regulations of development/ cell proliferation, proteins involved in the regulation of actin/cytoskeleton, and proteins involved in MAPK signaling pathways.In short, we identified the interacting proteins of PRAK by the combination of TAP system, DIGE and MS. Through this study, we draw the following conclusions:1. The PRAK coding region was cloned into TAP vector successfully. 2. The cell line stably-expressing TAP tagged-PRAK was established. TAP tags didn’t interfere with the localization of exogenous PRAK, and fusion protein didn’t change the antigenicity of CBP tag.3. TAP system was introduced successfully for purification of protein complexes. The cell line only expressing TAP tag stably was established successfully as the negative control for TAP system..4. By DIGE,14 differential protein spots between the HEK293-TAP-PRAK control group and the HEK293-TAP-PRAK-NaAsO2 group were found in PRAK protein complex obtained with TAP purification, and 12 candidate interacting proteins of PRAK were identified from the 14 differential protein spots by MS.