Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that interacts directly with MEK and MAPK. Here we show that KSR1 translocates from the cytoplasm to the cell surface in response to growth factor treatment and that this process is regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 constitutively associates with mammalian KSR1 and phosphorylates serine 392 to confer 14-3-3 binding and cytoplasmic sequestration of KSR1 in unstimulated cells. In response to signal activation, the phosphorylation state of S392 is reduced, allowing the KSR1 complex to colocalize with activated Ras and Raf-1 at the plasma membrane, thereby facilitating the phosphorylation reactions required for the activation of MEK and MAPK.

The physiological relevance of scaffolding proteins to MAPK activation was initially demonstrated in yeast, where Ste5 was shown to be required for activation of the MAPK module involved in pheromone mating [9, 10]. Summary Although a structural homolog of Ste5 has not been found in mammalian cells, proteins have been identified Background: Kinase Suppressor of Ras (KSR) is a conthat perform scaffolding functions for the ERK, JNK, served component of the Ras pathway that acts as a and p38 MAPK cascades [11-13]. Strikingly, a general molecular scaffold to facilitate signal transmission through feature that has emerged from studies of these scaffoldthe MAPK cascade. Although recruitment of KSR1 from ing proteins is their involvement in the spatiotemporal the cytosol to the plasma membrane is required for its regulation of MAPK activity. For example, JNK-interscaffolding function, the precise mechanism(s) regulatacting protein (JIP) scaffolds can direct JNK compoing the translocation of KSR1 have not been fully elucinents to specific subcellular regions of specialized cells, dated. such as growth cones of neurons, due to their ability Results: Using mass spectrometry to analyze the KSR1to be dynamically redistributed within cells by kinesin scaffolding complex, we identify the serine/threonine motor proteins [14]. In addition, the ␤-arrestins, which protein phosphatase PP2A as a KSR1-associated profunction as ERK and JNK3 scaffolds in G protein-coutein and show that PP2A is a critical regulator of KSR1 pled receptor pathways, retain active ERK and JNK3 in activity. We find that the enzymatic core subunits of the cytoplasm, thereby restricting their activity to cyto-PP2A (PR65A and catalytic C) constitutively associate plasmic substrates [15]. Another ERK scaffold, Mek with the N-terminal domain of KSR1, whereas binding partner 1 (MP1), localizes to late endosomes via an interof the regulatory PR55B subunit is induced by growth action with the p14 adaptor protein and plays a specialfactor treatment. Specific inhibition of PP2A activity preized role in ERK1 activation at this site [16]. Significantly, vents the growth factor-induced dephosphorylation ectopic targeting of the MP1/p14 complex to the plasma event involved in the membrane recruitment of KSR1 membrane prevents MP1-mediated augmentation of and blocks the activation of KSR1-associated MEK and ERK signaling. Thus, uncovering the mechanisms that ERK. Moreover, we find that PP2A activity is required regulate the intracellular localization of different scaffor activation of the Raf-1 kinase and that both Raf and folding proteins is critical for understanding their func-KSR1 must be dephosphorylated by PP2A on critical tion in MAPK activation. regulatory 14-3-3 binding sites for KSR1 to promote For the mammalian KSR1 scaffold, its intracellular MAPK pathway activation. localization is dynamic and changes in response to Ras Conclusions: These findings identify KSR1 as novel activation events. In quiescent cells, KSR1 is found in the substrate of PP2A and demonstra...

Osteoclast maturation is accompanied by changes in podosome patterning, resulting in the formation of a peripheral belt, which requires an intact microtubule network. Here, we report that by inhibiting Rho, the podosome belt is maintained at the cell periphery despite depolymerisation of microtubules by nocodazole. Rho inhibition was correlated to the increase in microtubule stabilisation and microtubule acetylation. By microinjecting activated Rho or its activated effector mDia2 in osteoclasts, we found that the podosome belt was disrupted and the level of microtubule acetylation dramatically decreased. We further characterised the molecular mechanism responsible for microtubule deacetylation by co-immunoprecipitation experiments. We found that not only was mDia2 coprecipitating with the recently identified microtubule deacetylase HDAC6 but that it also activated the microtubule deacetylase activity of HDAC6 in an in vitro deacetylase assay. Finally, we found that during osteoclastogenesis, there is a correlation between the increase in microtubule acetylation and the podosome belt stabilisation and that if Rho is inhibited in the early stages of osteoclast differentiation, it accelerates both microtubule acetylation and podosome belt stabilisation. Altogether, our data reveal a pathway in which Rho interferes with the osteoclast maturation process by controlling the level of microtubule acetylation and actin organisation through mDIA2 and HDAC6.

Efficient T cell activation requires at least two signals, one mediated by the engagement of the TCR-CD3 complex and another one mediated by a costimulatory molecule. We recently showed that CD46, a complement regulatory receptor for C3b as well as a receptor for several pathogens, could act as a potent costimulatory molecule for human T cells, highly promoting T cell proliferation. Indeed, we show in this study that CD46/CD3 costimulation induces a synergistic activation of extracellular signal-related kinase mitogen-activated protein kinase. Furthermore, whereas T lymphocytes primarily circulate within the bloodstream, activation may induce their migration toward secondary lymphoid organs or other tissues to encounter APCs or target cells. In this study, we show that CD46/CD3 costimulation also induces drastic morphological changes of primary human T cells, as well as actin relocalization. Moreover, we show that the GTP/GDP exchange factor Vav is phosphorylated upon CD46 stimulation alone, and that CD46/CD3 costimulation induces a synergistic increase of Vav phosphorylation. These results prompted us to investigate whether CD46/CD3 costimulation induced the activation of GTPases from the Rho family. Indeed, we report that the small GTPase Rac is also activated upon CD46/CD3 costimulation, whereas no change of Rho and Cdc42 activity could be detected. Therefore, CD46 costimulation profoundly affects T cell behavior, and these results provide important data concerning the biology of primary human T cells.

Cells from the myeloid lineage, namely macrophages, dendritic cells and osteoclasts develop podosomes instead of stress fibers and focal adhesions to adhere and migrate.Podosomes share many components with focal adhesions but differ in their molecular organization, with a dense core of polymerized actin surrounded by scaffolding proteins, kinases and integrins. Podosomes are found either isolated both in macrophages and dendritic cells or arranged into superstructures in osteoclasts. When osteoclasts resorb bone, they form an F-actin rich sealing zone, which is a dense array of connected podosomes that firmly anchors osteoclasts to bone. It delineates a compartment in which protons and proteases are secreted to dissolve and degrade the mineralized matrix. Since Rho GTPases have been shown to control F-actin stress fibers and focal adhesions in mesenchymal cells, the question of whether they could also control podosome formation and arrangement in cells from the myeloid lineage, and particularly in osteoclasts, rapidly emerged. This article considers recent advances made in our understanding of podosome arrangements in osteoclasts and how Rho GTPases may control it.

RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology.Introduction: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis. Materials and Methods: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest. Results: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts. Conclusions: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/ Wrch-1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N-and C-terminal domains. The N-terminal extension promotes binding to SH3 (Src homology 3)-domain-containing adaptors, whereas the C-terminal extension mediates membrane targeting through palmitoylation of its non-conventional CAAX box. RhoU/Wrch-1 possesses transforming activity, which is negatively regulated by its N-terminal extension and depends on palmitoylation.Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c-Srcexpressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N-terminal extension and the palmitoylation site were dispensable, whereas the C-terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked-down. Conclusions.Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C-terminal domains are critical for these functions.

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.