Scraped-Wounding Causes Activation and Association of C-Src Tyrosine Kinase with Microtubules in Cultured Keratinocytes.

Abstract

ABSTRACT. In order to elucidate the function of c-Src in keratinocytes, we studied the intracellular distribution of its active and inactive form in cultured normal human keratinocyte, using anti-c-Src monoclonal antibody clone 28, which recognizes the active form of c-Src (dephosphorylated at COOH-terminal residue Tyr 530), and monoclonal antibody clone 327 which recognizes both active and inactive forms. Since c-Src has been suggested to be involved in the control of cell adhesion in other cells, we produced a dynamic condition of cell migration by cutting culture cell colonies into squares to form a mesh pattern with a blade (culture wound model). Before cutting, the active form was expressed in cells located only at the periphery of colonies or isolated migrating cells, and was associated with microtubules. Wounding the colony generated a dramatic and rapid activation of c-Src in a few rows of cells along the cut edges, which were made even at the middle of colony, resulting in the association of the active form with microtubules. This increase of the active form was also detected by immunoblotting of cell extracts. These reactions were inhibited by 1 mM sodium orthovanadate, a protein-tyrosine phosphatase inhibitor. ST 638, a potent Src family tyrosine kinase inhibitor, inhibited the migration of keratinocytes in the culture wound healing model. These results suggest that wounding the culture causes activation of c-Src in keratinocytes, and thus activated c-Src may play a role in the function of microtubules during cell migration, especially at an early stage of wound healing.Key words: c-Src/keratinocyte/wound healing/microtubules During wound healing, keratinocytes (KCs) must dynamically regulate cell-cell contacts and cytoskeletons in order to migrate to and cover the surface of the wound. Major cell-cell contacts between KCs are adherens junctions and desmosomes, the former of which are specifically associated with actin microfilaments and the latter with keratin-intermediate filaments. The intercellular contacts of adherens junctions are mediated in a homotypic manner with Ecadherin (Takeichi, 1991), and linked to actin filaments via vinculin, =-actinin, =-catenin, >-catenin, plakoglobin and P120-Cas (Kam et al., 1995;Tsukita et al., 1992;Reynolds et al., 1992Reynolds et al., , 1994Reynolds et al., , 1996. Increase in tyrosine phosphorylation of >-catenin, plakoglobin and P120-Cas, which are known as substrate of c-Src, has been shown to be correlated with decrease of cell adhesion, which is one of the typical characteristics of neoplastic transformation (Matsuyoshi et al., 1992; Behrens et al., 1993;Hamaguchi et al., 1993;Papkoff, 1997). The regulation of cell-cell contacts is thought to be one of the most important events for KCs to cover the wound surface (Grinnell, 1990), and the following differentiation. Therefore, it is of potential importance to clarify the intracellular signaling mechanisms involved in wound healing in skin.Src is a non receptor type tyrosine kinase, which is widely expressed ...

Cdk5 regulates activation and localization of Src during corneal epithelial wound closure

“…Src is activated along the leading edge of scratch-wounded cultures of corneal epithelial cells and in corneal debridement wounds in organ-cultured eyes. A similar pattern of Src activation was previously observed in scratch-wounded keratinocyte cultures (Yamada et al, 2000). In accordance with previously reported functions of Src (Frame et al, 2002), Src activation in corneal epithelial cells is associated with dissolution of focal adhesions, remodeling of the actin cytoskeleton and formation of lamellipodia.…”

150thAnniversary Series: Desmosomes and Autoimmune Disease, Perspective of Dynamic Desmosome Remodeling and Its Impairments in Pemphigus

“…The dynamic weak-adhesive desmosomes, which lose their contacts already at 90 min after transferred to low Ca 2 ϩ -medium (Ca 2 ϩ -dependent; Wallis et al, 2000;Garrod et al, 2005;Kimura et al, 2007;Garrod & Kimura, 2008), are formed in both preconfl uent keratinocytes and keratinocytes at the edge of wounds in vitro, where EGFR and Src are activated for accelerating keratinocyte migration, rather than proliferation Yamada et al, 2000). On the other hand, the stable hyper-adhesive desmosomes are found in keratinocytes cultured longer than 6 days under confl uent culture conditions, which retained desmosomal contacts for several hours even after transferred to low Ca 2 ϩ -medium (Ca 2 ϩ -independent; Wallis et al, 2000;Garrod et al, 2005;Kimura et al, 2007;Garrod & Kimura, 2008).…”

Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment

“…MDia1 exerts it effects on actin and MTs through its FH1 and FH2 regions, respectively . Interestingly, inhibitors of the Src tyrosine kinase inhibit mDia1 function and wound closure (Owens et al, 2000;Satoh and Tominaga, 2001;Yamada et al, 2000). Additionally, mDia works in association with ROCK for the formation of stress fibers and focal adhesions (Nakano et al, 1999).…”