Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

Abstract

We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/ threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr 18 /Ser 19 -MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr 18 -and pSer 19 -MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr 18 -MRLC without any effect on nuclear pSer 19 -MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyaminedepleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.putrescine; ␣-difluoromethylornithine; F-actin; focal adhesion kinase; paxillin POLYAMINES [spermidine, spermine, and their diamine precursor putrescine (PUT)] are found in all tissues of almost all living species. These polyamines are organic, aliphatic, cationic amines. At physiological pH, they bind to proteins, DNA, RNA, and other negatively charged molecules to regulate diverse biological effects. The first rate-limiting step in polyamine synthesis is the production of PUT from the amino acid ornithine. This reaction is catalyzed by ornithine decarboxylase (ODC), which has one of the shortest half-lives of any mammalian enzyme and is present at extremely low levels in quiescent cells. Its activity is highly regulated to accommodate cellular function (6, 7). In our previous studies (17, 37, 46 -49), we showed that the early phase of mucosal healing due to cell migration requires polyamines and that polyamine depletion by means of treatment with ␣-difluoromethylornithine (DFMO), a specific inhibitor of ODC, inhibits migration in a rat stress ulcer model. Subsequently, we showed that the small intestinal epithelial crypt cell line IEC-6 could be used as an in vitro model to study the process of cell migration inv...

MLCK regulates Schwann cell cytoskeletal organization, differentiation and myelination

“…Orange ovals, SC nuclei; blue circles, axons; red lines, inhibition; blue arrow, activation. describing the presence of its endogenous targets (myosin II and/ or MLC) in the nucleus of human and rodent cells (Li and Sarna, 2009;Ray et al, 2007). MLCK activity has been shown to be necessary for TNFa-dependent NFkB activation and amplification of pro-survival signals (Wadgaonkar et al, 2003).…”

Polyamine-dependent activation of Rac1 is stimulated by focal adhesion-mediated Tiam1 activation

“…This assay was carried out as described previously. 35,36 Cells grown in control, DFMO, or DFMO plus putrescine containing media were serum starved for 24 h before the experiment. Six-well plates containing a confluent monolayer of cells were marked in the center (along the diameter) with a marker.…”

“…Rac1 activity was determined by pull down assay as described previously. 36,37 GST-PAK fusion protein bound with the glutathione agarose beads was mixed with cell lysate (200 µg). The binding was allowed to proceed for 1.5 h at 4°C, the beads were washed with lysis buffer and the amount of GTP-Rac1 bound was analyzed by SDS-PAGE and western blot using Rac1 specific antibody.…”

Myosin IIB deficiency in embryonic fibroblasts affects regulators and core members of the par polarity complex

“…Our and other results (Even-Ram et al 2007;Vidali et al 2006) have identified Rac-1 activation as one of these signals. This notion is supported by reports that assaulting the organization of the actin cytoskeleton alters Rac-1 activation/expression (Di Ciano et al 2002;Katsumi et al 2002;Vitale and Carbajal 2004;Ray et al 2007;Sharma and Mayer 2008;Desai et al 2008;Baumer et al 2008).…”