In this study, we show that the role of nonmuscle myosin II (NMII)-B in front–back migratory cell polarity is controlled by a short stretch of amino acids containing five serines (1935–1941). This motif resides near the junction between the C terminus helical and nonhelical tail domains. Removal of this motif inhibited NMII-B assembly, whereas its insertion into NMII-A endowed an NMII-B–like ability to generate large actomyosin bundles that determine the rear of the cell. Phosphomimetic mutation of the five serines also inhibited NMII-B assembly, rendering it unable to support front–back polarization. Mass spectrometric analysis showed that several of these serines are phosphorylated in live cells. Single-site mutagenesis showed that serine 1935 is a major regulatory site of NMII-B function. These data reveal a novel regulatory mechanism of NMII in polarized migrating cells by identifying a key molecular determinant that confers NMII isoform functional specificity.

Intermediate filament (IF) attachment to intercellular junctions is required for skin and heart integrity, but how the strength and dynamics of this attachment are modulated during normal and pathological remodeling is poorly understood. We show that glycogen synthase kinase 3 (GSK3) and protein arginine methyltransferase 1 (PRMT-1) cooperate to orchestrate a series of posttranslational modifications on the IF-anchoring protein desmoplakin (DP) that play an essential role in coordinating cytoskeletal dynamics and cellular adhesion. Front-end electron transfer dissociation mass spectrometry analyses of DP revealed six novel serine phosphorylation sites dependent on GSK3 signaling and four novel arginine methylation sites including R2834, the mutation of which has been associated with arrhythmogenic cardiomyopathy (AC). Inhibition of GSK3 or PRMT-1 or overexpression of the AC-associated mutant R2834H enhanced DP–IF associations and delayed junction assembly. R2834H blocked the GSK3 phosphorylation cascade and reduced DP–GSK3 interactions in cultured keratinocytes and in the hearts of transgenic R2834H DP mice. Interference with this regulatory machinery may contribute to skin and heart diseases.

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.

The first step in the assembly of new chromatin is the cell cycle–regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

The assembly of the vessel wall from its cellular and extracellular matrix components is an essential event in embryogenesis. Recently, we used the descending aorta of the embryonic quail to define the morphological events that initiate the formation of a multilayered vessel wall from a nascent endothelial cell tube (Hungerford, J.E., G.K. Owens, W.S. Argraves, and C.D. Little. 1996. Dev. Biol. 178:375–392). We generated an mAb, 1E12, that specifically labels smooth muscle cells from the early stages of development to adulthood. The goal of our present study was to characterize further the 1E12 antigen using both cytological and biochemical methods. The 1E12 antigen colocalizes with the actin cytoskeleton in smooth muscle cells grown on planar substrates in vitro; in contrast, embryonic vascular smooth muscle cells in situ contain 1E12 antigen that is distributed in threadlike filaments and in cytoplasmic rosette-like patterns. Initial biochemical analysis shows that the 1E12 mAb recognizes a protein, M r = 100,000, in lysates of adult avian gizzard. An additional polypeptide band, M r = 40,000, is also recognized in preparations of lysate, when stronger extraction conditions are used. We have identified the 100-kD polypeptide as smooth muscle α-actinin by tandem mass spectroscopy analysis. The 1E12 antibody is an IgM isotype. To prepare a more convenient 1E12 immunoreagent, we constructed a single chain antibody (sFv) using recombinant protein technology. The sFv recognizes a single 100-kD protein in gizzard lysates. Additionally, the recombinant antibody recognizes purified smooth muscle α-actinin. Our results suggest that the 1E12 antigen is a member of the α-actinin family of cytoskeletal proteins; furthermore, the onset of its expression defines a primordial cell restricted to the smooth muscle lineage.