HHBRK1,a Small Novel Protein, Associates With Cardiac Troponin T And Enriches At The Protruding Lamellipodia In Living Cells

The actin cytoskeleton is a dynamic structure that plays an important role in cell shape, cell migration events and spatial orgnaization of signaling. Cell motility is initiated via the controlled nucleation and polymerization of actin filaments to produce sheet- or finger-like projections, termed lamellipodia and filopodia that are induced, respectively, via activation of the Rho family members, Rac 1 and Cdc42. Disruption of actin filaments and a decrease in focal adhensions are common features of transformed cells. The alterations in actin filament structure were found to correlate with decreased or upcreased expression of various cytoskeleton proteins.BRK1, a small novel protein, is thought to be involved in actin-dependent aspects of cell polarization. Mutation of brk1 blocks the enrichments of cortical F-actin at the tips of emerging lobes in the maize leaf. A novel gene that is up regulated in human lung cancer tissues has been identified using a suppressive-subtractive hybridization. This gene, designated hHBrk1 (human homology of Brick1), encodes a small protein hHBRK1, which is highly conserved throughout the mammalian and plant kingdom. Computer assisted analysis of hHBRK1 amino acid sequences unveiled a highly conserved protein domain that is characterized by a heptad repeat (HR) motif with a potential for α-helical coiled coil formation.Ectopically expressed GFP-hHBRKl fusion protein distributed diffusely in the cell cytoplasm and becomes enriched at the leading edge of living cells. During cell division, it remains its global cortical association and is enriched in the actomyosin ring. N-terminal deletion derivative resulted in diffusion pattern within the cell. While the C-terminal deletion derivative in which the HR motif was deleted, GFP fusion protein diffused all over the cell, notably, it's also enriched to Golgi complex. The mutation derivatives in which the HR motifs were disrupted didn't affect the hHBRKl special subcellular location pattern. These results suggested that hHBRKl is involved in actin nucleation, and hHBRKl-cTnT interaction may regulate actin cytoskeketal organization. HR motif plays a role in dynamic equilibrium of hHBRKl protein recruitment to Golgi complex.The binding partner of hHBRKl was detected in heart tissues of mouse. The interaction between hHBRKl and cardiac troponin T (cTnT) was unveiled by GST pull-down assay and confirmed by coimmunoprecipitation. A similar coiled coil domain is also conserved in cTnT amino termini sequences. The evolutionary conserved HR motif may play a role in hHBRKl-cTnT dimerization, presumably through the formation of ct-helical coiled coils. There results indicate that hHBRKl might associate with cardiac troponin T to regulate actin organization.Cardiac troponin T, a thin filament myofibrillar protein, is essential for the Ca + regulation of cardiac muscle contraction in vertebrates. The N-terminalregion of cTnT is particularly significant because it is a site for alternative splicing between the many different cTnT isoforms expressed in cardiac muscle. A similar potentially coiled coil-forming motif is also conserved in all known cTnT tail domain. These protein motifs appear to be the regions where hHBRKl-cTnT interaction might take place.