Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCKactomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116Rip , whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116Rip , through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116Rip inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116Rip is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.

p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen. Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown. Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serumdeprived and LPA-stimulated cells, with the N terminus (residues 1-382) dictating cytoskeletal localization. In addition, p116Rip is found in the nucleus. Direct interaction or colocalization with RhoA was not detected. We find that p116Rip binds tightly to F-actin (K d ϳ 0 .5 M) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II. Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy. When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actinbinding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation. Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.

Various factors are involved in the heat shock-induced inhibition of protein synthesis. Changes upon heat shock in phosphorylation, leading to inactivation, of eukaryotic initiation factors (eIFs) eIF2 and eIF4E have been shown for several cell types. However, in mammalian cells these changes occur at temperatures of 43°C or higher while protein synthesis is already affected at milder heat shock temperatures. In searching for the cause for the inhibition of protein synthesis, the regulation of eIF2 and eIF4E by additional factors was analyzed. In this respect, the activity of eIF2B was measured during and after heat shock. A very clear correlation was found between the activity of this guanine exchange factor and the levels of protein synthesis, also at mild heat shock conditions. Changes in the phosphorylation of eIF4E and of the eIF4E-binding protein PHAS-I were also analyzed. Surprisingly, in H35 cells as well as in some other cell lines, PHAS-I phosphorylation was increased by heat shock, whereas in others it was decreased. Therefore, decreasing the eIF4E availability under stressful conditions does not seem to be a general mechanism to inhibit protein synthesis by heat shock. Regulation of eIF2B activity appears to be the main mechanism to control translation initiation after heat shock at mild temperatures.Incubating mammalian cells at elevated temperatures inhibits the translation of mRNAs (1-4). The translational lesion in heat-shocked cells principally occurs at the initiation step of translation (1,(5)(6)(7) for review, see Ref. 8). In the initiation process, the 7-methyl guanosine cap at the 5Ј end of the mRNA is bound by a complex of eukaryotic initiation factors (eIFs), 1 including the cap-binding protein eIF4E and the RNA helicase eIF4A. Unwinding of the RNA enables recognition of the AUG initiation codon by the 43 S ribosomal complex and delivery of initiator methionyl tRNA. Then the large ribosomal subunit joins the complex, and peptide synthesis begins (8).Both the concentration and activity of protein synthesis initiation factors and the primary sequence of the 5Ј-untranslated region of an mRNA can affect the rates of eukaryotic translation initiation. With respect to the heat shock-induced modifications of eukaryotic protein synthesis initiation factors, it is of interest that the activity of many initiation factors can be regulated by phosphorylation. It has been shown that changes in the phosphorylation state of various of these eukaryotic initiation factors, such as eIF2, eIF2B, eIF4B, and eIF4E, could coincide with changes in protein synthesis (9, 10).The proteins eIF2 and eIF2B act to bring Met-tRNA to the 40 S ribosomal subunit: eIF2 by binding to Met-tRNA and GTP, and eIF2B by replacing GDP on eIF2 for GTP. Previous studies from several laboratories have focused on heat shock-induced phosphorylation of eIF2␣ (11-13). However, it was found that less severe heat shocks that inhibit protein synthesis by more than 70% do not elicit this phosphorylation. Duncan and Hershey (13) suggested tha...

The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini.