Identification Of The Interaction Between Guanine Nucleotide Exchange Factor Vavl And MINT In Nuclear Matrix

MINT (Msx2-interacting nuclear target protein) is a nuclear matrix protein originally cloned as an interacting protein of MSX2,a homeodomain transcription repressor functioning in the craniofacial skeletal and neural development.MINT belongs to the Spen (split end) protein family which plays an essential role in multiple developmental events.Spen proteins vary in a wide range in size (90-600 kD),but are nevertheless characterized by a conserved domain structure,indluding three repeated RNA recognition motifs(RRMs) near the N terminus and a conserved SPOC (Spen paralog and ortholog C-terminal domain) domain at the C terminus, which mediates interaction with the SMRT/N-CoR corepressors. The human homolog of MINT, SHARP ( SMRT/HDAC1-associated repressor protein ), has been identified as a component in transcriptional repression complexes recruited by nuclear receptors.The MINT/SHARP-mediated repression was sensitive to the HDAC (Histone deacetylases) inhibitor TSA,and SHARP is a novelcomponent of the HDAC corepressor complex,suggesting that MINT/SHARP represses transcription in an HDAC-dependent fashion.Recently,Kuroda et al and Oswald et al demonstrated that MINT and SHARP also interacts with RBP-J (Recombination signal binding protein-Jk ) ,a key transcription factor downstream of Notch recrptor,and represses the RBP-J-mediated transactivation by competing for the binding site and by recruiting co-repressors including SMRT/N-CoR and HDAC,and may thus regulate the Notch signaling pathway.Kuroda et al also showed that targeted disruption of MINT lead to embryonic lethality,with a developmental retardation of multiple organs.However,as a widely expressed protein,possible functions mechanism of MINT remain to be revealed.In the beginning,to study the status and function of MINT in developmental signaling,yeast two-hybrid assay was performed to screen proteins the can interact with fragment 1 of MINT from human lymph cDNA library and got a significant molecule:Vavl.The Vav family of guanine nucleotide exchange factors (GEFs) represent a critical link between antigen receptor-coupled protein-tyrosine kinases and the signaling pathways controlled by the Rho family of GTPases. Vav GEFs are highly homologous proteins composed of a catalytic Dbl homologous (DH) domain, a pleckstrin homology (PH) domain, one Src homology 2 (SH2) domain, and two SH3 domains. In mammalian although Vavl is mostly restricted to hematopoietic cells, Vav2 and Vav3 display a much broader tissue expression.Vavl are tyrosine-phosphorylated through the stimulation of diverse receptors, including the epidermal growth factor receptors, the platelet-derivedgrowth factor receptors, integrins, and B and T cell antigen receptors (BCRs and TCRs).After engagement of the antigen receptor, the IT AMs ( Immunoreceptor tyrosine-based activation motifs) are tyrosine phosphorylated by activated Src-family protein-tyrosine kinases (PTKs). The phosphorylated IT AMs serve as docking sites for the tandem Src-homology 2 (SH2)-domain-containing PTKs SYK and ZAP70 . The activation of SYK and ZAP70 leads to the phosphorylation of several molecules, which, in turn, activate signaling networks . Several tyrosine-phosphorylated molecules have been shown to interact with the SH2 domain of Vavl, including the adaptor proteins SLP76 (SH2-domain-containing leukocyte protein of 76 kDa) in T cells and BLNK (B-cell linker ; SLP65/BASH) in B cells. Co-receptors, such as CD28 on T cells and CD 19 on B cells, also participate in the activation of Vavl.The recruitment of Vavl into large molecular scaffolds regulate several cell processes,such as activation of the MAPK pathway and gene activation, including NFAT ,NF- k B , and AP-1 . Vavl as key components of the antigen receptor signal transduction machinery by integration of signals that control the cell cycle , differentiation ,cytoskeleton rearrangement to form immunological synapses and apoptosis.In the present study,we furtherly use the yeast two-hybrid assay to study the interaction between MINT and Vavl.The bait plasmid was condtructed by cloning MINT-Fx(X represents 1-6) into the vector pGBKT7, several different fragments of the C-terminal of Vavl based on the Vav's different domains were subcloned into the vector pGADT7 or pACT2 to test the interaction with MINT-Fl and their interaction was tested by the growth of yeast assay.Then we got the results that only the MINT-F1 can interact with Vavl,and the exact...