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 Lplastin 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. ' 2007 Wiley-Liss, Inc.Key words: migration; invasion; adhesion; L-plastin; melanoma Malignant melanoma is a very aggressive type of cancer that frequently metastasizes to distant organs. The capacity of tumor cells to form metastatic foci correlates with the ability to proteolytically degrade basement membrane barriers and to adhere to and migrate through extracellular matrix layers. 1,2 Two classes of molecules are mainly involved in these processes: the matrix metalloproteinases (MMPs) 3 and integrins. 4,5 Integrins are a large family of adhesion receptors involved in cell-cell and cellextracellular matrix interactions. They consist of heterodimers of 2 noncovalently associated a-and b-subunits. 6 The role of integrins in tumor cell metastasis has been intensively studied. 4,5,7 In human melanoma, especially b1-and b3-subunit containing integrins play important roles in metastasis, through an enhancement of adhesion, migration and invasion. 8,9 Moreover, the cell surface expression levels of many integrins can be directly correlated with the metastatic potential of melanoma cells. [10][11][12] Modulation of the actin cytoskeleton is critical f...

Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane-associated antigen critical to niche homing

“…23,26,27 We confirm this pathogenic role using an in vivo xenotransplantation model of leukemia. Finally, we demonstrate that BCR-targeted therapies may deactivate LCP1, thereby blocking migratory behavior, a mechanism of action consistent with clinical observations of peripheral lymphocytosis using these inhibitors.…”

Ectopic expression of L-plastin in human tumor cells: Diagnostic and therapeutic implications

“…MV3 cell populations stably expressing wt-L-plastin or non-phosphorylatable 5A7A-L-plastin showed no change in static adhesion to collagen I and fibronectin. However, in a haptotactic migration assay, both wt-L-plastin and 5A7A-L-plastin expressing cells migrated faster towards collagen I and fibronectin than the control cells (Klemke et al, 2007). Thus, L-plastin enhances haptotactic migration independent of its phosphorylation state.…”

“…Interestingly, with regard to tumor cell invasiveness, wt-L-plastin expressing melanoma cells behaved differently from cells expressing the non-phosphorylatable 5A7A-L-plastin. Although 5A7A-L-plastin expressing cells and control cells were to a certain extent able to invade the Matrigel layer, an up to threefold increase occurred, if cells expressed phosphorylatable wt-L-plastin (Klemke et al, 2007). Thus, not only expression but also phosphorylation of L-plastin is required to enhance tumor cell invasiveness.…”

“…However, L-plastin expression did not correlate with tumor stages or penetration depths (Klemke et al, 2007). Still, esiRNA-mediated knock-down of L-plastin in a human melanoma cell line negatively influenced cell migration.…”

“…1) were changed to non-phosphorylatable Ala (5A7A-Lplastin). Using 2D-gel-electrophoresis we first showed that in MV3 cells wt-L-plastin undergoes spontaneous phosphorylation, which was not observed in cells expressing 5A7A-L-plastin (Klemke et al, 2007). MV3 cell populations stably expressing wt-L-plastin or non-phosphorylatable 5A7A-L-plastin showed no change in static adhesion to collagen I and fibronectin.…”