Multisite Phosphorylation of the Guanine Nucleotide Exchange Factor Cdc24 during Yeast Cell Polarization

BACKGROUND:Cell polarization is essential for processes such as cell migration and asymmetric cell division. A common regulator of cell polarization in most eukaryotic cells is the conserved Rho GTPase, Cdc42. In budding yeast, Cdc42 is activated by a single guanine nucleotide exchange factor, Cdc24. The mechanistic details of Cdc24 activation at the onset of yeast cell polarization are unclear. Previous studies have suggested an important role for phosphorylation of Cdc24, which may regulate activity or function of the protein, representing a key step in the symmetry breaking process. METHODOLOGY/PRINCIPAL FINDINGS:Here, we directly ask whether multisite phosphorylation of Cdc24 plays a role in its regulation. We identify through mass spectrometry analysis over thirty putative in vivo phosphorylation sites. We first focus on sites matching consensus sequences for cyclin-dependent and p21-activated kinases, two kinase families that have been previously shown to phosphorylate Cdc24. Through site-directed mutagenesis, yeast genetics, and light and fluorescence microscopy, we show that nonphosphorylatable mutations of these consensus sites do not lead to any detectable consequences on growth rate, morphology, kinetics of polarization, or localization of the mutant protein. We do, however, observe a change in the mobility shift of mutant Cdc24 proteins on SDS-PAGE, suggesting that we have indeed perturbed its phosphorylation. Finally, we show that mutation of all identified phosphorylation sites does not cause observable defects in growth rate or morphology. CONCLUSIONS/SIGNIFICANCE:We conclude that lack of phosphorylation on Cdc24 has no overt functional consequences in budding yeast. Yeast cell polarization may be more tightly regulated by inactivation of Cdc42 by GTPase activating proteins or by alternative methods of Cdc24 regulation, such as conformational changes or oligomerization

Physiology: Relax and come in

During inflammation, lymph nodes swell with an influx of immune cells. New findings identify a signalling pathway that induces relaxation in the contractile cells that give structure to these organs

Langerin+ dendritic cells are responsible for LPS-induced reactivation of allergen-specific Th2 responses in postasthmatic mice.

International audienceAllergic asthma is a T cell-dependent inflammatory lung disease that results from complex interactions between genetic predisposition and environmental factors, including exposure to lipopolysaccharide (LPS). In this study, we have shown that airway LPS exposure was sufficient to induce airway hyperreactivity (AHR) and eosinophil recruitment in mice that had previously experienced an acute episode of allergic asthma. LPS-induced disease reactivation depended on the activation of allergen-specific CD4(+) T cells by a subset of lung langerin(+) dendritic cells (DCs) that retained the allergen. Upon LPS exposure, migration of langerin(+) DCs from lungs to draining lymph nodes increased and LPS-exposed langerin(+) DCs instructed CD4(+) T cells toward a T helper (Th) 2 response. Selective depletion of langerin(+) DCs prevented LPS-induced eosinophil recruitment and T-cell activation, further demonstrating a critical role for langerin(+) DCs in disease reactivation. This finding provides a possible explanation for the subclinical worsening of asthmatics following exposure to low-dose LPS

N-terminal Dbl domain of the RhoGEF, Kalirin.

Guanine nucleotide exchange factors (GEF) promote the release of GDP from GTPases, thus allowing the free GTPase molecule to bind the more abundant GTP molecule. In the GTP-bound state, the GTPase elicits signal transduction by acting on its effector proteins. Spontaneous release of GDP is a slow process and the catalysis of the GDP release by a GEF is generally a prerequisite for efficient signaling (Vetter and Wittinghofer 2001). The structurally related GEFs form subfamilies that regulate a specific family of GTPase proteins. GEFs that activate Rho GTPases have been implicated in cancer and mental retardation. RhoGEFs are a relatively large family, and many of the *69 human RhoGEFs were discovered based on their oncogenic activation in cancer and cancer models. The catalytic components of RhoGEFs are referred to as Dbl homology domains, after the screen that identified the protein Dbl encoded by the diffuse B-cell lymphoma (dbl) oncogene (Eva and Aaronson 1985). Thus the RhoGEF family is a potential target for treating tumors and cancer

The Salmonella Typhimurium effector SteC inhibits Cdc42-mediated signaling through binding to the exchange factor Cdc24 in Saccharomyces cerevisiae.

Intracellular survival of Salmonella relies on the activity of proteins translocated into the host cell by type III secretion systems (T3SS). The protein kinase activity of the T3SS effector SteC is required for F-actin remodeling in host cells, although no SteC target has been identified so far. Here we show that expression of the N-terminal non-kinase domain of SteC down-regulates the mating and HOG pathways in Saccharomyces cerevisiae. Epistasis analyses using constitutively active components of these pathways indicate that SteC inhibits signaling at the level of the GTPase Cdc42. We demonstrate that SteC interacts through its N-terminal domain with the catalytic domain of Cdc24, the sole S. cerevisiae Cdc42 guanine nucleotide exchange factor (GEF). SteC also binds to the human Cdc24-like GEF protein Vav1. Moreover, expression of human Cdc42 suppresses growth inhibition caused by SteC. Of interest, the N-terminal SteC domain alters Cdc24 cellular localization, preventing its nuclear accumulation. These data reveal a novel functional domain within SteC, raising the possibility that this effector could also target GTPase function in mammalian cells. Our results also highlight the key role of the Cdc42 switch in yeast mating and HOG pathways and provide a new tool to study the functional consequences of Cdc24 localization