We report a novel mechanism concerning the differential phosphorylation of fascin in response to cell-matrix adhesion conditions. We have used biochemical, pharmacological, and molecular genetic approaches to establish that α5β1 integrin–mediated adhesion to fibronectin correlates with stimulation of fascin phosphorylation and that the molecular mechanism involves a PKC-dependent process in which serine 39 of fascin is the required site for phosphorylation. Adhesion to TSP-1 or laminin-1, conditions under which cells assemble stable microspikes or ruffles containing fascin and F-actin, does not stimulate fascin phosphorylation. Furthermore, the ability of cells to regulate fascin phosphorylation and thereby their actin-binding activity is of functional significance in matrix attachment, spreading, and cytoskeletal organization under different matrix-adhesion conditions.
Fascin was discovered as an actin-bundling protein in sea urchin egg extracts. Species orthologues in Drosophila
and in mammalian cells have also been demonstrated to bind and bundle actin into tightly packed, highly ordered arrays (reviewed by Edwards and Bryan, 1995
). More recently, the localization of fascin to cell surface spikes and projections at the leading edges of migratory mammalian cells and of fascin-transfected cells has suggested a major role in the formation of cellular protrusions (Tao et al., 1996
; Adams, 1997
; Yamashiro et al., 1998
). Several independent analyses have indeed indicated that the assembly of fascin spikes or projections is of functional significance for cell motile behavior (Adams, 1997
; Yamashiro et al., 1998
). Fascin-containing projections may also participate in other cellular activities. The antigen-presentation interactions of dendritic cells with T-cells involve the formation of close cell-to-cell appositions that are mediated by the finger-like dendritic projections of dendritic cells. Treatment of epidermal Langerhans cells with fascin antisense oligonucleotides inhibits the formation of these dendrites (Ross et al., 1998
). The presence of fascin-rich spikes and membrane projections on neuronal growth cones may indicate a role in axon guidance or adhesion (Edwards and Bryan, 1995
; Adams, unpublished observations).
It has been established that cell adhesion to specific extracellular matrix macromolecules provides potent regulation of fascin distribution and microspike formation (Adams, 1995
). On fibronectin, fascin-containing projections are formed transiently during the initial, postattachment spreading and are rapidly lost as cells adopt a polygonal, spread morphology. Fascin then appears uniformly diffuse. On TSP-1, fascin microspikes are formed in large arrays and remain stably adherent during cell spreading (Adams, 1995
). The data presented here identify a biochemical mechanism that underlies these correlative effects. Adhesion to fibronectin mediated by α5β1 integrin leads to PKCα-dependent phosphorylation of fascin at serine 39. This down-regulates the ability of fascin to bind and bundle actin (Ono et al., 1997
). In the absence of PKCα, these events do not take place, and spikes and actin-associated fascin are retained at later times of adhesion. Interestingly, LLC-PK1 cells expressing the nonphosphorylatable fascin S39A formed many small fascin-containing projections when attached on fibronectin but were impaired in cell spreading. Yet, cells expressing fascin S39D spread more extensively than cells expressing wild-type fascin. Thus, the ability to modulate the actin-binding activity of fascin through phosphorylation appears to be an important required step in cell adhesion and focal contact assembly on fibronectin. Our data demonstrate that phosphorylation at this site is an in vivo target of PKCα activity, because PKCα down-regulation strongly altered the distribution of wild-type fascin yet did not markedly affect the distribution of fascin S39A or S39D. LLC-PK1 cells contain little endogenous fascin and provide a low background for observation of the effects of mutant fascins.
The experiments also demonstrate that adhesion of C2C12 cells on TSP-1 does not lead to phosphorylation of fascin. This suggested a possible mechanism for the stable nonpolarized formation of microspikes, in that fascin remains competent to bind actin in spread cells. This hypothesis was tested by expression of the mutant fascins in C2C12 cells. C2C12 cells expressing fascin S39A spread and formed arrays of spikes that appeared disorganized compared with the spikes of control C2C12 cells, whereas C2C12 cells expressing fascin S39D remained round. Thus, nonphosphorylated fascin is required for cells to form microspikes on TSP-1. The ability to cycle fascin phosphorylation could be important for the initial organization of adherent fascin spikes when cells contact TSP-1; however, because the activities of the mutant fascins in C2C12 cells are displayed against a large pool of endogenous fascin, it was not possible to define this point further in this experimental system.
The lack of specific colocalization of fascin with actin bundles or microfilaments in fibronectin-adherent cells raises the possibility that fascin interacts with other protein(s) under these conditions. A reported second binding partner is β-catenin (Tao et al., 1996
). We were unable to obtain evidence for an increase in the amount of fascin binding to β-catenin in fibronectin-adherent or TPA-treated cells. Interactions with skeletal muscle tropomyosin reversibly inhibit fascin-actin binding, and it is possible that such interactions may be dominant for phosphorylated fascin, which has low affinity for actin (Ishikawa et al., 1998
). Alternatively, fascin may have additional noncytoskeletal binding partners.
Activation of PKCα is recognized as an early signaling event consequent to α5β1 integrin–mediated attachment to fibronectin and has also been correlated with ligation of the vitronectin-binding integrins αvβ3 and αIIb/β3 (Vuori and Ruoslahti, 1993
; Lewis et al., 1995
; reviewed by Kolanus and Seed, 1997
). PKCα localizes to focal contacts in long-term adherent cells and preferentially interacts with active β1 integrins (Jaken et al., 1989
; Ng et al., 1999
). Stimulation or inhibition of PKC, respectively, promotes or inhibits the organization of focal adhesions in prespread cells (Woods and Couchman, 1992
). Similarly, activation of PKC by TPA or overexpression of a constitutively activated PKCα mutant correlates with enhanced cell spreading and motility (Vuori and Ruoslahti, 1993
; Rigot et al., 1998
; Miranti et al., 1999
; Sun and Rotenburg, 1999
). Cells in which PKC is inhibited pharmacologically or down-regulated typically show reduced spreading (Chun and Jacobson, 1993
; Gao et al., 1996
; Miranti et al., 1999
). We have obtained similar results in C2C12 cells. The dramatically increased spreading on fibronectin of GFP-fascin expressor LLC-PK1 cells in which PKCα is down-regulated is thus an unusual and surprising response. To our knowledge, this is the first examination of the effects of PKCα down-regulation on the matrix adhesion of these kidney epithelial cells. One possible explanation could be that secondary effects of PKCα down-regulation on other regulatory molecules or elements of the cytoskeleton result in an abnormal state of cell contraction. Indeed, certain mRNAs are stabilized in LLC-PK1 cells upon down-regulation of PKC (Nanbu et al., 1994
Several components of the submembranous cytoskeleton are substrates of PKC; these include profilin (Hansson et al., 1988
), MARCKS, an actin-binding protein that functions in initial spreading on fibronectin (Myat et al., 1997
), and the focal contact components vinculin and talin (Werth et al., 1983
; Werth and Pastan, 1984
; Beckerle, 1990
; reviewed by Jaken, 1996
). The mechanism by which PKC promotes focal contact assembly may involve stabilization of interactions between the integrin cytoplasmic domain and talin (Burn et al., 1988
; Woods and Couchman, 1992
). In addition, phosphorylation of vinculin tail domain by PKC is increased in the presence of acidic phospholipids. It has been established that acidic phospholipids inhibit the intramolecular interactions of vinculin head and tail domains, thereby exposing the actin- and paxillin-binding sites. Phosphorylation of unfolded vinculin tail domain by PKC may further facilitate its incorporation into focal contacts. This activity may underlie the poor formation of focal contacts by cells in which PKCα is down-regulated or inhibited by blockade of fibronectin binding to α5β1 integrin (Schweinbacher et al., 1996
; Weekes et al., 1996
; Huttelmaier et al., 1998
). Further experiments will examine whether such regulation of the vinculin-actin interaction contributes indirectly to the regulation of microspike formation by extracellular matrix.
Such promotion of focal contact organization by activated PKC contrasts with its inhibitory effects on actin and/or fascin spikes. We postulate that cell adhesion on TSP-1 may involve low activity of PKC in the cell cortex. Evidence in support of this view is provided by (a) the absence of phosphorylated fascin in TSP-1–adherent cells; (b) the loss of microspikes from TSP-1–adherent cells upon strong activation of PKCα by phorbol ester; and (c) localization studies that indicate that PKCα is not detectable in the cortical regions of TSP-1–adherent cells. However, the impairment of adhesion to TSP-1 in cells treated with PKCα pseudosubstrate peptide is suggestive of a role for PKCα in the early stages of attachment. A likely scenario may be that PKCα is transiently or weakly activated when cells attach to TSP-1 and may be needed to phosphorylate substrates that play a role in receptor clustering or actin nucleation. In the absence of sustained PKCα activity in the cell cortex, cross-linking of actin by fascin would take place and lead to microspike formation. Indeed, in another experimental system, TSP-1 has been found to activate distinct signaling events involving a Gi-type heterotrimeric G protein (Gao et al., 1996
). Alternatively, PKC activity may form part of a parallel pathway that can be overridden in the context of adhesion to TSP-1. One interesting possibility is that adhesion to TSP-1 may activate a serine phosphatase. These possibilities will be addressed in further experiments.
Cell-staining studies have shown that fascin projections and microspikes are not uniformly present on all cells under standard tissue culture conditions. When formed, the projections tend to be restricted to localized domains of the cell surface (Yamashiro-Matsumura and Matsumura, 1986
; Adams, 1995
; Tao et al., 1996
). These observations imply the existence of physiological mechanisms that modulate the formation of fascin spikes and projections in conjunction with other adhesive contacts such as focal contacts. Experimental 1:1 mixed fibronectin/TSP-1 substrata stimulate concurrent formation of focal contacts and fascin spikes, and this phenomenon depends on both integrins and proteoglycans (Adams, 1997
). Here we have established a molecular mechanism, namely the promotion or inhibition of PKC-dependent fascin phosphorylation, by which cell adhesion to specific extracellular matrix macromolecules differentially regulates the ability of cells to assemble fascin projections. In the context of a complex tissue extracellular matrix, specific microenvironments may either facilitate or disfavor the localized or polarized formation of fascin spikes and filopodia and thereby modulate cell-adhesive and migratory behavior.