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PDZ-LIM domain proteins and α-actinin at the muscle Z-diskKlaavuniemi, T. (Tuula) 24 November 2006 (has links)
Abstract
The Z-disk is a sophisticated structure that connects adjacent sarcomeres in striated muscle myofibrils. α-Actinin provides strength to the Z-disks by crosslinking the actin filaments of adjacent sarcomeres. α-Actinin is an antiparallel homodimer, composed of an N-terminal actin binding domain (ABD), the central rod domain, and two pairs of C-terminal EF-hands. The PDZ-LIM domain proteins interact with α-actinin at the Z-disk. Of these proteins, only the actinin-associated LIM protein (ALP), Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) and C-terminal LIM protein (CLP36) have a ZASP/Cypher-like (ZM) motif consisting of 26-27 conserved residues in the internal region between the PDZ and LIM domains. The aim of this work was to understand the molecular interplay between the ZM-motif containing members of the PDZ-LIM proteins and α-actinin. To unveil the biological relevance of the interaction between the PDZ-LIM proteins and α-actinin, naturally occurring human ZASP/Cypher mutations were analyzed.
Two interaction sites were found between ALP, CLP36 and α-actinin using recombinant purified proteins in surface plasmon resonance (SPR) analysis. The PDZ domain of ALP and CLP36 recognized the C-terminus of α-actinin, whereas the internal regions bound to the rod domain. Further characterization showed that the ALP internal region adopts and extended conformation when interacting with α-actinin and that the ZM-motif partly mediated the interaction, but did not define the entire interaction area. ZASP/Cypher also interacted and competed with ALP in binding to the rod domain. The internal fragments containing the ZM-motif were important for co-localization of ALP and ZASP/Cypher with α-actinin at the Z-disks and on stress fibers. The absence of ALP and ZASP/Cypher in focal contacts indicates that other interacting molecules, for instance vinculin and integrin, may compete in binding to the rod in these areas or additional proteins are required in targeting to these locations. The co-localization of the ZASP/Cypher with α-actinin could be released by disrupting the stress fibers leading to an accumulation of α-actinin in the cell periphery, whereas ZASP/Cypher was not in these areas. This suggests that an intact cytoskeleton is important for ZASP/Cypher interaction with α-actinin. Earlier studies have shown that mutations in the ZASP/Cypher internal region are associated with muscular diseases. These mutations, however, did not affect ZASP/Cypher co-localization with α-actinin or the stability of ZASP/Cypher proteins.
The Z-disk possesses a stretch sensor, which is involved in triggering hypertrophic growth as a compensatory mechanism to increased workloads. α-Actinin is a docking site of molecules that are involved in hypertrophic signaling cascades mediated by calsarcin-calcineurin and protein kinase C (PKC) isoforms. The internal interaction site may be involved in targeting PKCs, which bind to the LIM domains of ZASP/Cypher, to the Z-disks. The similar location of the internal interaction site with calsarcin on the rod suggests that ZASP/Cypher, ALP and CLP36 may regulate calsarcin-mediated hypertrophic signaling.
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Tension-Dependent Formation of Stress Fibers in Fibroblasts : A Study Using Semi-Intact CellsHIRATA, Hiroaki, TATSUMI, Hitoshi, SOKABE, Masahiro 12 1900 (has links)
No description available.
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The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial CellsHolmqvist, Kristina January 2004 (has links)
<p>Angiogenesis is defined as the formation of new capillary blood vessels from pre-existing ones. This process involves several steps including: migration, proliferation and differentiation of endothelial cells into blood vessels. Angiogenesis is initiated by binding of specific growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to their cell surface receptors. Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors. My aim has been to identify the role of Shb in FGF- and VEGF-signalling in endothelial cells. Shb was found to be phosphorylated in a Src-dependent manner upon both FGF- and VEGF-stimulation. This was confirmed using fibroblasts overexpressing temperature sensitive v-Src. Furthermore, Shb-induced cell spreading on collagen of immortalised brain endothelial (IBE) cells was also Src-dependent. FGF stimulation led to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding domain of Shb. IBE cells overexpressing wild-type or R522K Shb (inactive SH2 domain) displayed increased FAK activation on collagen.</p><p>The SH2-domain of Shb was found to bind to tyrosine 1175 in the VEGFR-2 in a phosphotyrosine dependent manner using PAE cells expressing VEGFR-2. Furthermore, by use of siRNA, Shb knock-down experiments revealed that Shb regulates FAK activity, cellular migration and stress fiber formation in response to VEGF stimulation of VEGFR-2. In summary, Shb binds to both FGFR-1 and VEGFR-2 and regulates the activity of FAK and thereby stress fiber formation and cellular migration, which are necessary for formation of new blood vessels. IBE cells with an inactive SH2 domain of Shb displayed disorganised formation of tubular structures in the tube formation assay, while overexpression of wild-type Shb led to accelerated tubular morphogenesis.</p><p>Taken together, my data show that the adaptor protein Shb plays an important role in the process angiogenesis, in response to angiogenic tyrosine kinase receptors, by interacting with FAK and regulating spreading, stress fiber formation and cellular migration.</p>
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The Role of Shb in Angiogenesis, FGF and VEGF Signalling in Endothelial CellsHolmqvist, Kristina January 2004 (has links)
Angiogenesis is defined as the formation of new capillary blood vessels from pre-existing ones. This process involves several steps including: migration, proliferation and differentiation of endothelial cells into blood vessels. Angiogenesis is initiated by binding of specific growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), to their cell surface receptors. Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors. My aim has been to identify the role of Shb in FGF- and VEGF-signalling in endothelial cells. Shb was found to be phosphorylated in a Src-dependent manner upon both FGF- and VEGF-stimulation. This was confirmed using fibroblasts overexpressing temperature sensitive v-Src. Furthermore, Shb-induced cell spreading on collagen of immortalised brain endothelial (IBE) cells was also Src-dependent. FGF stimulation led to a direct association between Shb and FAK, which was mediated by the phosphotyrosine binding domain of Shb. IBE cells overexpressing wild-type or R522K Shb (inactive SH2 domain) displayed increased FAK activation on collagen. The SH2-domain of Shb was found to bind to tyrosine 1175 in the VEGFR-2 in a phosphotyrosine dependent manner using PAE cells expressing VEGFR-2. Furthermore, by use of siRNA, Shb knock-down experiments revealed that Shb regulates FAK activity, cellular migration and stress fiber formation in response to VEGF stimulation of VEGFR-2. In summary, Shb binds to both FGFR-1 and VEGFR-2 and regulates the activity of FAK and thereby stress fiber formation and cellular migration, which are necessary for formation of new blood vessels. IBE cells with an inactive SH2 domain of Shb displayed disorganised formation of tubular structures in the tube formation assay, while overexpression of wild-type Shb led to accelerated tubular morphogenesis. Taken together, my data show that the adaptor protein Shb plays an important role in the process angiogenesis, in response to angiogenic tyrosine kinase receptors, by interacting with FAK and regulating spreading, stress fiber formation and cellular migration.
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