Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells.
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 polyamine-depleted 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.
Ray
Ramesh M
RM
Department of Physiology, The University of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163, USA. rray@physio1.utmem.edu
Guo
Huazhang
H
Patel
Minesh
M
Jin
Shi
S
Bhattacharya
Sujoy
S
Johnson
Leonard R
LR
D000199:Actins / Q000378:metabolism; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1 / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism*; D051419:Paxillin / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011073:Polyamines / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D054648:Protein Phosphatase 2; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology*; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D020830:rac1 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000199:Actins; D000634:Aminoquinolines; D018994:Myosin Light Chains; C487513:NSC 23766; D051419:Paxillin; D011073:Polyamines; D047428:Protein Kinase Inhibitors; C496063:Pxn protein, rat; D011743:Pyrimidines; D051417:Focal Adhesion Kinase 1; C496052:Ptk2 protein, rat; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein; D019311:Staurosporine