title: Phosphorylation of ectopically expressed L-plastin enhances invasiveness of human melanoma cells.
abstract: The leukocyte specific actin-binding protein L-plastin is aberrantly expressed in several nonhematopoetic malignant tumors. However, little is known about the functional consequences of L-plastin expression. Here, we investigated the function of L-plastin in human malignant melanoma cells. Knock-down of endogenous L-plastin by siRNA treatment reduced migration of the melanoma cell line IF6. However, in melanoma patients, no correlation existed between L-plastin expression and tumor stages. This implied that additional factors such as phosphorylation of L-plastin may influence its function in tumor cells. To investigate this further, EGFP-tagged wild-type L-plastin (wt-LPL-EGFP) and a mutated, nonphosphorylatable L-plastin protein (5A7A-LPL-EGFP), were expressed in the L-plastin negative melanoma cell line MV3. Biochemical analysis revealed that wt-LPL-EGFP is phosphorylated in MV3 cells while 5A7A-LPL-EGFP is not. Although both wt-LPL-EGFP and 5A7A-LPL-EGFP were targeted to, and promote the formation of, vinculin-containing adhesion sites, static adhesion to either Matrigel or isolated extracellular matrix molecules was neither influenced by expression of wt-LPL-EGFP nor by expression of 5A7A-LPL-EGFP when compared with EGFP expressing control cells. In contrast, haptotactic, but not chemotactic, migration of melanoma cells towards either Matrigel or isolated extracellular matrix molecules was similarly enhanced, if either 5A7A-LPL-EGFP or wt-LPL-EGFP were expressed in MV3 cells. Interestingly, only cells expressing the phosphorylatable wt-LPL-EGFP protein showed enhanced invasion into Matrigel. In line with these findings the in vivo metastatic capacity of mouse B16 melanoma cells correlates with expression and phosphorylation of L-plastin. These data show that an increase in melanoma cell invasiveness requires not only expression but also phosphorylation of L-plastin.
authors.affiliation: Department of Immunology and Serology, University of Heidelberg, D-69120 Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Klemke
authors.forename: Maria T
authors.initials: MT
authors.lastname: Rafael
authors.forename: Guido H
authors.initials: GH
authors.lastname: Wabnitz
authors.forename: Tatjana
authors.initials: T
authors.lastname: Weschenfelder
authors.forename: Mathias H
authors.initials: MH
authors.lastname: Konstandin
authors.forename: Natalio
authors.initials: N
authors.lastname: Garbi
authors.forename: Frank
authors.initials: F
authors.lastname: Autschbach
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hartschuh
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Samstag
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D003094:Collagen / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004338:Drug Combinations; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007797:Laminin / Q000378:metabolism; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins; D051379:Mice; D008840:Microfilament Proteins; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009367:Neoplasm Staging; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D011509:Proteoglycans / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D016596:Vinculin / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D004338:Drug Combinations; D016326:Extracellular Matrix Proteins; D007797:Laminin; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D011509:Proteoglycans; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C026742:plastin; C059485:matrigel; D016596:Vinculin; D049452:Green Fluorescent Proteins; D003094:Collagen