Coupling S100A4 to Rhotekin alters Rho signaling output in breast cancer cells.

Rho signaling is increasingly recognized to contribute to invasion and metastasis. In this study, we discovered that metastasis-associated protein S100A4 interacts with the Rho-binding domain (RBD) of Rhotekin, thus connecting S100A4 to the Rho pathway. Glutathione S-transferase pull-down and immunoprecipitation assays demonstrated that S100A4 specifically and directly binds to Rhotekin RBD, but not the other Rho effector RBDs. S100A4 binding to Rhotekin is calcium-dependent and uses residues distinct from those bound by active Rho. Interestingly, we found that S100A4 and Rhotekin can form a complex with active RhoA. Using RNA interference, we determined that suppression of both S100A4 and Rhotekin leads to loss of Rho-dependent membrane ruffling in response to epidermal growth factor, an increase in contractile F-actin 'stress' fibers and blocks invasive growth in three-dimensional culture. Accordingly, our data suggest that interaction of S100A4 and Rhotekin permits S100A4 to complex with RhoA and switch Rho function from stress fiber formation to membrane ruffling to confer an invasive phenotype.

Phosphoinositide 3-kinase signaling is critical for ErbB3-driven breast cancer cell motility and metastasis.

Many malignancies show increased expression of the epidermal growth factor (EGF) receptor family member ErbB3 (HER3). ErbB3 binds heregulin ß-1 (HRGß1) and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGF receptor (EGFR; HER1), enhancing phosphorylation of specific C-terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of phosphoinositide 3 (PI3)-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGß1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGß1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, whereas mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor.

DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation.

DAP-kinase (DAPk) is a Ser/Thr kinase that regulates cytoplasmic changes associated with programmed cell death. It is shown here that a GFP-DAPk fusion, which partially localized to actin stress fibers, induced extensive membrane protrusions. This phenotype correlated with changes in myosin-II distribution and with increased phosphorylation of the myosin-II regulatory light chain (RLC). A mutant lacking the cytoskeletal-interacting region (GFP-DAPkDeltaCyto) displayed diffuse cytoplasmic localization, and induced peripheral membrane blebbing, instead of the extensive protrusions. In contrast, deletion of the ankyrin repeats led to mislocalization of the kinase to focal contacts, where it failed to elicit any changes in cell morphology. While both wild-type DAPk and DAPkDeltaCyto induced RLC phosphorylation independently of the Rho-activated kinase ROCK, only the wild type led to increases in stress-fiber associated phospho-RLC. Thus, the precise intracellular localization of DAPk is critical for exposure to its substrates, including the RLC, which mediate varying morphologic changes.

Role of myosin-II phosphorylation in V12Cdc42-mediated disruption of Drosophila cellularization.

Microinjection of constitutively active Cdc42 (V12Cdc42) disrupts the actomyosin cytoskeleton during cellularization (Crawford et al., Dev. Biol., 204, 151-164 (1998)). The p21-activated kinase (PAK) family of Ser/Thr kinases are effectors of GTP-bound forms of the small GTPases, Cdc42 and Rac. Drosophila PAK, which colocalizes with actin and myosin-II during cellularization, concentrates at sites of V12Cdc42-induced actomyosin disruption. In vitro biochemical analyses demonstrate that PAK phosphorylates the regulatory light chain (RLC) of Drosophila nonmuscle myosin-II on Ser21, a site known to activate myosin-II function. Although activated PAK does not disrupt the actomyosin cytoskeleton, it induces increased levels of Ser21 phosphorylated RLC. These findings suggest that increased levels of RLC phosphorylation do not contribute to disruption of the actomyosin hexagonal array.

Differential regulation of alternatively spliced endothelial cell myosin light chain kinase isoforms by p60(Src).

The Ca(2+)/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V(max), and a 2-fold increase in K(0.5 CaM) when compared with the SM MLCK isoform, whereas K(m) was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK). This increased activity of phospho-MLCK-1 was observed over a broad range of submaximal [Ca(2+)] levels with comparable EC(50) [Ca(2+)] for both phosphorylated and unphosphorylated EC MLCK-1. The sites of tyrosine phosphorylation catalyzed by p60(Src) are Tyr(464) and Tyr(471) within the 69-residue stretch deleted in the MLCK-2 splice variant. These results demonstrate for the first time that p60(Src)-mediated tyrosine phosphorylation represents an important mechanism for splice variant-specific regulation of nonmuscle MLCK and vascular cell function.

Protein kinase C mediates phosphorylation of the regulatory light chain of myosin-II during mitosis.

Phosphorylation of the regulatory light chain (RLC) of myosin-II is cell cycle dependent. Early in mitosis the RLC is phosphorylated predominantly on Ser-1/2, while during cytokinesis the primary site of phosphorylation is Ser-19 (Yamakita et al., 1994). To identify candidate kinases likely to mediate the mitotic phosphorylation on Ser-1/2, we assayed RLC kinase activity in mitotic cell extracts and measured apparent steady-state kinetic constants using purified enzymes. The mitotic RLC kinase is distinct from cdc2 kinase, protein kinase A and protein kinase G, as activators or inhibitors specific for these kinases do not affect the mitotic kinase activity. The activity of the mitotic RLC kinase is enhanced by the addition of Ca2+ and DAG and/or phorbol esters, characteristics of a conventional protein kinase C (PKC). Moreover, the PKC inhibitors, Gö6983 and Gö6976, significantly attenuate the phosphorylation of the RLC in mitotic extracts. Apparent steady-state kinetic studies indicate that several PKC isoforms display high specificity for myosin-II. These results suggest that current models describing Ser-1/2 phosphorylation during mitosis need to be re-evaluated.

Localization and activity of myosin light chain kinase isoforms during the cell cycle.

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.

Molecular mechanisms of nonmuscle myosin-II regulation.

Myosin II, the conventional two-headed myosin that forms bipolar filaments, is directly involved in regulating cytokinesis, cell motility and cell morphology in nonmuscle cells. To understand the mechanisms by which nonmuscle myosin-II regulates these processes, investigators are now looking at the regulation of this molecule in vertebrate nonmuscle cells. The identification of multiple isoforms of nonmuscle myosin-II, whose activities and regulation differ from that of smooth muscle myosin-II, suggests that, in addition to regulatory light chain phosphorylation, other regulatory mechanisms control vertebrate nonmuscle myosin-II activity.

Structure determination and characterization of Saccharomyces cerevisiae profilin.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

A subset of protein kinase C phosphorylation sites on the myosin II regulatory light chain inhibits phosphorylation by myosin light chain kinase.

Protein kinase C (PKC) phosphorylates the regulatory light chains of smooth muscle and cytoplasmic myosin II at three known sites: S1, S2, and T9 [Ikebe, M., Hartshorne, D. J., & Elzinga, M. (1987) J. Biol. Chem. 262, 9569-9573]. Phosphorylation at these sites inhibits the actomyosin ATPase and inhibits phosphorylation of S19 on the regulatory light chain by myosin light chain kinase (MLCK) [Nishikawa, M., Sellers, J. R., Adelstein, R. S., & Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814]. To compare the effects of phosphorylation at a subset of PKC sites on the rate of MLCK phosphorylation, we substituted alanines for the known PKC phosphorylation sites in the Xenopus regulatory light chain (XRLC). PKC phosphorylation of S1A/S2A/T9A revealed secondary phosphorylation sites at T7 and T10, which are accessible both on isolated S1A/S2A/T9A and S1A/S2A/T9A-myosin hybrids. Apparent kinetic constants were determined for MLCK phosphorylation of WT XRLC and XRLC mutants: T9A, S1A/S2A, S1A/S2A/T9A, and T7A/T9A/T10A. PKC prephosphorylation of S1/2 had no effect on the rate of MLCK phosphorylation, while PKC prephosphorylation of T7/9/10 inhibited MLCK phosphorylation due to a 6-fold increase in Km. Our results suggest that phosphorylation of RLC S1/2 as observed in vivo may not be responsible for an inhibition of MLCK phosphorylation.

Phosphorylation on threonine-18 of the regulatory light chain dissociates the ATPase and motor properties of smooth muscle myosin II.

We cloned the full-length cDNA for the cytoplasmic myosin II regulatory light chain (RLC) from a stage 1-2 Xenopus oocyte library. The Xenopus RLC is 94% identical to the chicken smooth muscle myosin RLC. All of the protein kinase C and myosin light chain kinase phosphorylation sites are conserved. Using trifluoperazine [Trybus, K. M., Waller, G. S., & Chatman, T. A. (1994) J. Cell Biol. 124, 963-969], we removed the RLC of smooth muscle myosin and replaced it with recombinant Xenopus RLCs. The wild-type Xenopus RLC substitutes for the gizzard RLC in actin-activated ATPase and in vitro motility assays. We made alanine substitutions of the two residues phosphorylated by myosin light chain kinase, Ser-19 and Thr-18. All of the myosin hybrids, regardless of their mutations or phosphorylation, have similar K+EDTA ATPase activities. As expected, the T18A, S19A hybrid had no actin-activated ATPase, whereas the T18A hybrid phosphorylated on Ser-19 had an actin-activated ATPase similar to that of wild-type hybrids phosphorylated only on Ser-19. The actin-activated ATPase of myosin phosphorylated only on Thr-18 is approximately 15-fold lower than that of myosin phosphorylated on Ser-19. Phosphorylation of either Ser-19 or Thr-18 permits the formation of filaments. Remarkably, in the gliding filament assay, myosin phosphorylated only on Thr-18 moves actin filaments at velocities similar to myosin phosphorylated on Ser-19 or both Thr-18 and Ser-19.

Evidence that a 27-residue sequence is the actin-binding site of ABP-120.

Proteolysis experiments of ABP-120 from Dictyostelium discoideum have previously demonstrated that removal of residues 89-115 from a tryptic peptide which retains actin binding activity, abolishes actin binding (Bresnick, A. R., Warren, V., and Condeelis, J. (1990) J. Biol. Chem. 265, 9236-9240). Antibodies made against a synthetic peptide of this 27-amino acid sequence (27-mer) specifically immunoprecipitate native ABP-120 from Dictyostelium high speed supernatants, demonstrating that the 27-mer sequence is on the surface of the molecule as expected for an active site. ABP-120 is inhibited in its binding to F-actin by Fab' fragments of the anti-27-mer IgG. Half-maximal inhibition occurs at an approximate molar ratio of 7 Fab' fragments/ABP-120 monomer. Viscoelastic measurements indicate that ABP-120 forms fewer cross-links with F-actin in the presence of the 27-mer synthetic peptide than in its absence. In F-actin cosedimentation assays, the binding of ABP-120 to actin is inhibited by the 27-mer synthetic peptide. Furthermore, the 27-mer synthetic peptide cosediments with F-actin, whereas a control hydrophobic peptide and a synthetic peptide of residues 69-88 of ABP-120 do not cosediment with F-actin. These observations suggest a direct involvement of the 27-mer sequence in the actin binding activity of ABP-120.

Identification of a short sequence essential for actin binding by Dictyostelium ABP-120.

Tryptic digestion of ABP-120, an actin cross-linking protein from Dictyostelium discoideum, generates a ladder of peptides differing in molecular mass by 13,000 daltons, indicating a structural repeat within the molecule. A number of peptides bind actin with the smallest having a molecular mass of 17,000 daltons (T17). Our sedimentation assays also show that a peptide of 14,000 daltons does not bind actin. Using the full-length cDNA sequence (Noegel, A., Rapp, S., Lottspeich, F., Schleicher, M., and Stewart, M. (1989) J. Cell Biol. 109, 607-618) and protein sequencing techniques, we have determined that T17 begins at residue 89 while T14 begins at residue 116. Therefore we have localized 27 amino acids which are essential for actin binding activity. This region is at the end of the molecule, distal from the repetitive beta-sheet region predicted from the cDNA sequence, and displays high sequence identity with regions in the N termini of ABP/filamin, dystrophin, beta-spectrin, and alpha-actinin.