Primary Study The Expression Of F-actin Ralated Protein HHBrk1 In Tumor And The Interaction With Myosin â…¥

Proper execution of wide variety of actin-dependent processes, including the cell division and motility, depend on tight control over the timing and location of actin polymerization. It is well established that Rho-GTPase induces actin based plasma membrane projections by activating the Arp2/3 complex via WASP/ Scar /WAVE proteins. hHBrk1, also be denominated as C3orf10, MDS027 and HSPC300, has been shown to interact with the N-terminal Scar homology domains (SHD) of WAVE protein. The association of HSPC300/ hHBrk1 with Scar/WAVE might play a role in WAVE activation, localization and stabilization. We previous showed that hHBrk1 protein contains 75 amino acids, with a conserved heptads repeat motif (HR) at the C terminus and specifically recruits to the tips of lamellipodia and actomyosin ring. Mutation at HR domain affects its subcellular location and hHBrk1Δ47-75 deletion variant recruits notably to Golgi complex.Here we report the characterization of the binding partners of hHBRK1 and the involvement of hHBRK1 in carcinogenesis. First, the expression profiling of hHBrk1 gene in human tumors and matched normal tissues was detected by Northern blot on cancer profiling array. HHBrk1 gene was expressed in all of the tissues and cell lines, as compared with the matched normal tissues, significantly higher expression level of hHBrk1 was detected in lung cancer tissues, and inversely lower expressed level was found in kidney cancer tissues. The following RT-PCR essay in 8 pairs of lung cancer tissues and matched normal tissues further confirmed the result. These results suggested that hHBrk1 gene might involve in the tumorigenesis and progress of lung cancer.Second, binding partners of hHBRK1 was scanned in liver tissue of mouse by GST pull-down assay followed peptide finger mass spectrometry. The coding region of hHBrk1 was subcloned into plasmid pGEX4T, fused in frame to generate GST-hHBRK1. GST-hHBRK1 fusion protein was purified from bacterial lysates using glutathiol Sepharose 4B affinity chromatography. The interaction between hHBRK1 and myosin VI was unveiled by GST pull-down assay and further confirmed by co-immunoprecipitation. We also observed the co-localization of hHBRK1 and myosin VI in endochylema of 95D cells, a human lung cancer cell line with high metastasis potency by confocal laser scanning microscopy.Last, the truncated isoform of hHBRK1 and myosin VI was constructed and expressed in order to characterize the binding sites between hHBRK1 and myosin VI. The binding sites in myosin VI are located to the coiled-coil domain and globular domain. While the binding site in hHBRK1 is located to the 47-69th amino acids residues as detected by co-immunoprecipitation.