Cell interaction with adhesive proteins or growth factors in the extracellular matrix initiates Ras/ mitogen-activated protein (MAP) kinase signaling. Evidence is provided that MAP kinase (ERK1 and ERK2) influences the cells' motility machinery by phosphorylating and, thereby, enhancing myosin light chain kinase (MLCK) activity leading to phosphorylation of myosin light chains (MLC). Inhibition of MAP kinase activity causes decreased MLCK function, MLC phosphorylation, and cell migration on extracellular matrix proteins. In contrast, expression of mutationally active MAP kinase kinase causes activation of MAP kinase leading to phosphorylation of MLCK and MLC and enhanced cell migration. In vitro results support these findings since ERK-phosphorylated MLCK has an increased capacity to phosphorylate MLC and shows increased sensitivity to calmodulin. Thus, we define a signaling pathway directly downstream of MAP kinase, influencing cell migration on the extracellular matrix.

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Death-associated protein kinase (DAPK) is a multidomain Ser/Thr protein kinase with an important role in apoptosis regulation. In these studies we have identified a DAPK-interacting protein called DIP-1, which is a novel multi-RING finger protein. The RING finger motifs of DIP-1 have E3 ligase activity that can auto-ubiquitinate DIP-1 in vitro. In vivo, DIP-1 is detected as a polyubiquitinated protein, suggesting that the intracellular levels of DIP-1 are regulated by the ubiquitin-proteasome system. Transient expression of DIP-1 in HeLa cells antagonizes the anti-apoptotic function of DAPK to promote a caspase-dependent apoptosis. These studies also demonstrate that DAPK is an in vitro and in vivo target for ubiquitination by DIP-1, thereby providing a mechanism by which DAPK activities can be regulated through proteasomal degradation.Regulation of protein degradation by the ubiquitin proteasome pathway is now known to be a major pathway through which cells modulate the expression levels of critical signaling proteins (1-6). This tightly regulated, complex pathway is a key regulator of many important signaling pathways and has an important role in many cellular processes including apoptosis, and recent studies have identified many apoptosis regulatory proteins as targets for ubiquitination (7-11). In addition to being targets for degradation, some apoptosis regulatory proteins have a more active role and act as components of the ubiquitin cascade via the ubiquitin ligase activity ascribed to the RING finger domains that is part of their primary structure. Targeting proteins for degradation by the ubiquitin proteasome pathway involves the covalent linkage of ubiquitin either to the amino terminus or specific lysine residues in the target protein through the action of three enzymes. In this process ubiquitin is first activated by an E1 ubiquitin-activating enzyme, transferred to an E2 ubiquitin-conjugating enzyme, and then ligated to the target protein by an E3 ubiquitin ligase (4,12) Recently the Ser/Thr protein kinase, death-associated protein kinase (DAPK) 1 has been implicated in apoptosis regulation. DAPK has a complex, multi-domain structure that includes a calcium/calmodulin-regulated kinase domain, a series of ankyrin repeats, and a carboxyl-terminal death domain (13-17). Although some of the regulatory features that directly control the catalytic activities of DAPK have been described, including the activation by calcium/calmodulin (17, 18) and the presence of an inhibitory autophosphorylation site (19), an understanding of how the cellular activities of DAPK are regulated in vivo is poorly understood. The presence of proteinprotein interaction domains within the primary structure of DAPK, including its ankyrin repeat motifs and death domain, suggests that additional interactions between DAPK and other cellular proteins will also be important for regulation of DAPK activities. In this study, we describe a new DAPK-interacting protein called DIP-1 (DAPK-interacting protein-1), which has a direct role in r...

Phosphorylation of myosin II regulatory light chains (RLC) by Ca2؉ /calmodulin-dependent myosin light chain kinase (MLCK) is a critical step in the initiation of smooth muscle and non-muscle cell contraction. Posttranslational modifications to MLCK down-regulate enzyme activity, suppressing RLC phosphorylation, myosin II activation, and tension development. Here we report that PAK2, a member of the Rho family of GTPasedependent kinases, regulates isometric tension development and myosin II RLC phosphorylation in saponin permeabilized endothelial monolayers. PAK2 blunts tension development by 75% while inhibiting diphosphorylation of myosin II RLC. Cdc42-activated placenta and recombinant, constitutively active PAK2 phosphorylate MLCK in vitro with a stoichiometry of 1.71 ؎ 0.21 mol of PO 4 /mol of MLCK. This phosphorylation inhibits MLCK phosphorylation of myosin II RLC. PAK2 catalyzes MLCK phosphorylation on serine residues 439 and 991. Binding calmodulin to MLCK blocks phosphorylation of Ser-991 by PAK2. These results demonstrate that PAK2 can directly phosphorylate MLCK, inhibiting its activity and limiting the development of isometric tension.The PAK 1 family of serine/threonine protein kinases have been implicated in a broad spectrum of signal transduction pathways leading to diverse physiological end points, including cytoskeletal reorganization, apoptosis, and Ras-mediated cell transformation (1, 2). All PAK isoforms are direct effectors of the Rho family GTP-binding proteins Rac and Cdc42, suggesting that cell-specific responses arise from either selective regulation of G protein activation in response to unique agonists or selective phosphorylation of tissue-specific protein kinase substrates.In the initial studies of the relative roles of G protein activation and protein kinase activity in actin reorganization, Sells et al. (3) demonstrated that microinjection of activated PAK1 induced rapid formation of polarized filopodia in Swiss 3T3 cells. However, the relative importance of PAK-dependent phosphorylation of unique substrates during Cdc42/Racdependent cytoskeletal reorganization is incompletely understood. Transient transfection of HeLa cells with constitutively active PAK1 promoted cytoskeletal rearrangement, which was entirely analogous to that observed in Cdc42 and Rac transfected cells (4). In subsequent studies, a peptide that inhibited kinase activity in PAK blocked Cdc42, Rac, and constitutively active PAK-induced morphological changes in transfected HeLa cells (5). An important role for PAK-mediated phosphorylation in actin reorganization has also been supported by studies in permeabilized (6) and intact (7) endothelial cells as well as skinned smooth muscle cells (8). Permeabilized endothelial cells incubated with Cdc42-activated PAK2 or the catalytic domain of PAK2 underwent ATP-dependent retraction and actin reorganization (6). In addition, these studies using purified enzymes established that the regulatory nonmuscle and smooth muscle myosin II light chain is a substrate for PAK2 (9, 1...

Novel E. coli mutants deficient in biosynthesis of 5- methylaminomethyl -2-thiouridine were isolated based on a phenotype of reduced readthrough at UAG codons. They define 2 new loci trmE and trmF , near 83' on the E. coli map. These mutants are different from strains carrying trmC mutations, which are known to confer a methylation deficiency in biosynthesis of 5- methylaminomethyl -2-thiouridine. tRNA from mutants carrying trmE or trmF mutations was shown to carry 2-thiouridine instead of 5- methylaminomethyl -2-thiouridine. This deficiency affects the triplet binding properties of the mutant tRNA. Our results suggest that the 5- methylaminomethyl group stabilizes the basepairing of this modified nucleotide with G, most likely through direct interaction with the ribosomal binding site(s).

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.

Circulating leukocytes are nonadherent but bind tightly to endothelial cells following activation. The increased avidity of leukocyte integrins for endothelial ligands following activation is regulated, in part, by interaction of the ␤2 subunit cytoplasmic tail with the actin cytoskeleton. We propose a mechanism to explain how tethering of the actin cytoskeleton to leukocyte integrins is regulated. In resting leukocytes, ␤2 integrins are constitutively linked to the actin cytoskeleton via the protein talin. Activation of cells induces proteolysis of talin and dissociation from the ␤2 tail. This phase is transient, however, and is followed by reattachment of actin filaments to integrins that is mediated by the protein ␣-actinin. The association of ␣-actinin with integrins may stabilize the cytoskeleton and promote firm adhesion to and migration across the endothelium. Glutathione S-transferase-␤2 tail fusion protein/mutagenesis experiments suggest that the affinity of ␣-actinin binding to the ␤2 tail is regulated by a change in the conformation of the tail that unmasks a cryptic ␣-actinin binding domain. Positive and inhibitory domains within the ␤2 tail regulate ␣-actinin binding: a single 11-amino acid region (residues 736 -746) is necessary and sufficient for ␣-actinin binding, and a regulatory domain between residues 748 -762 inhibits constitutive association of the ␤2 tail with ␣-actinin.Integrins are heterodimeric, transmembrane adhesion molecules composed of noncovalently associated ␣ and ␤ subunits that physically link extracellular ligands to the cytoskeleton (1). The cytoplasmic domain of integrin ␤ subunits links these receptors to the actin cytoskeleton (2, 3). However, actin filaments cannot bind directly to integrins. Instead, integrins are linked indirectly to actin filaments via several actin-binding proteins, including ␣-actinin, talin, and filamin. (4 -7). The importance of integrin-cytoskeletal linkage is demonstrated by the observation that deletion of the ␤ subunit tail prevents association of integrins with the cytoskeleton and disrupts normal integrin-ligand interactions (reviewed in Ref. 8).Several integrins, including LFA-1 1 and Mac-1, share a common ␤2 subunit and are present exclusively on leukocytes. These leukocyte integrins mediate cell adhesion to endothelial cell ligands such as intracellular adhesion molecules (reviewed in Ref. 9). Unactivated leukocytes in the circulation are nonadherent, and LFA-1 and Mac-1, both of which are expressed constitutively on resting neutrophils, show very little, if any, binding to their physiologic ligands (10). Activation of neutrophils with cytokines (leukotriene B 4 and tumor necrosis factor-␣), chemoattractants (FMLP), or phorbol 12-myristate 13-acetate (PMA) results in increased binding of neutrophils to the endothelium (11,12). This increase results, in part, from insertion of integrins onto the cell surface, but both LFA-1 and Mac-1 also undergo a rapid change in ligand avidity as a result of activation, which involves conformational changes...