Non-redundant roles of the Gab1 and Gab2 scaffolding adapters in VEGF-mediated signalling, migration, and survival of endothelial cells.

Gab1 was previously described as a positive modulator of Akt, Src, ERK1/2, endothelial cell migration, and capillary formation in response to vascular endothelial growth factor (VEGF). However, its involvement in endothelial cell survival, as well as the potential contribution of the other family member Gab2 to signalling and biological responses remained unknown. Here, we show that Gab2 is tyrosine phosphorylated in a Grb2-dependent manner downstream of activated VEGF receptor-2 (VEGFR2), and that it associates with signalling proteins including PI3K and SHP2, but apparently not with the receptor. Similarly to Gab1, over-expression of Gab2 induces endothelial cell migration in response to VEGF, whereas its depletion using siRNAs results in its reduction. Importantly, depletion of both Gab1 and Gab2 leads to an even greater inhibition of VEGF-induced cell migration. However, contrary to what has been reported for Gab1, the silencing of Gab2 results in increased Src, Akt and ERK1/2 activation, slightly reduced p38 phosphorylation, and up-regulation of Gab1 protein levels. Accordingly, re-expression of Gab2 in Gab2-/- fibroblasts leads to opposite results, suggesting that the modulation of both Gab2 and Gab1 expression in these conditions might contribute to the impaired signalling observed. Consistent with their opposite roles on Akt, the depletion of Gab1, but not of Gab2, results in reduced FOXO1 phosphorylation and VEGF-mediated endothelial cell survival. Mutation of VEGFR2 Y801 and Y1214, which abrogates the phosphorylation of Gab1, also correlates with inhibition of Akt. Altogether, these results underscore the non-redundant and essential roles of Gab1 and Gab2 in endothelial cells, and suggest major contributions of these proteins during in vivo angiogenesis.

Members of the plant NIMA-related kinases are involved in organ development and vascularization in poplar, Arabidopsis and rice.

NIMA-related kinases (Neks) are a family of serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals. Information regarding the function of Neks in plants is very limited. We screened the three plant species that have had their genomes sequenced in an attempt to improve our understanding of their role in plants. We retrieved seven members in Arabidopsis thaliana, nine in Populus trichocarpa and six in Oryza sativa. Phylogenetic analysis showed that plant Neks are closely related to each other and contain paralogous genes. Moreover, their chromosome distribution and their exon-intron structure revealed that the actual plant Nek family was derived from a single representative followed by large segmental duplication events. Functional expression analyses in the three species relied on RTqPCR in poplar and publicly available microarray data for Arabidopsis and rice. Although plant Neks are present in every organ analyzed, their expression profiles suggest their involvement in plant development processes. Furthermore, we showed that PNek1, a member of the poplar family, is expressed at sites of free auxin synthesis and is specifically involved during the vascularization process.

The scaffolding adapter Gab1 mediates vascular endothelial growth factor signaling and is required for endothelial cell migration and capillary formation.

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

Characterization of a poplar NIMA-related kinase PNek1 and its potential role in meristematic activity.

Meristems are sites of undifferentiated cell division, which carry on developing into functional organs. Using the two-hybrid system with a poplar 14-3-3, we uncovered poplar NIMA-related kinase 1 (PNek1) as an interacting protein. PNek1 shows high homology to the mammalian NIMA-related kinases, which are thought to be involved in cell cycle progression. Using a synchronized poplar cell suspension, we observed an accumulation of PNek1 mRNA at the G1/S transition and throughout the G2-to-M progression. Moreover, PNek1-GFP fusion protein localized in the cytoplasm and in both the nuclear and nucleolar regions. Overexpression of PNek1-GFP in Arabidopsis caused morphological abnormalities in flower and siliques. Overall, these results suggest that PNek1 is involved in plant development.

The two isoforms of the cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans.

We have cloned the Candida albicans TPK2 gene encoding a cAMP-dependent protein kinase (PKA) catalytic subunit and generated a tpk2 homozygous null mutant to assess its ability to germinate in liquid media. N-acetylglucosamine (GlcNAc)-induced germ-tube formation was attenuated in the tpk2 strain and enhanced by compounds that are known to increase the PKA activity in situ. Germination was completely blocked in the presence of the myristoylated derivative of the heat-stable PKA inhibitor (MyrPKI). These results indicate that TPK1 acts positively in regulating the morphogenetic transition in C. albicans in the absence of the TPK2 gene. We were able to identify an mRNA from this second form of PKA in both wild-type and tpk2 null mutant cells. We found that PKA activity measured in the mutant lacking the TPK2 gene was about 10% of that displayed by the wild-type. The finding that the germinative response of tpk2 null mutant to serum was severely diminished at low serum concentrations indicates that the level of PKA is an important determinant of filamentous growth at low serum concentrations. The extent of germination attained at higher serum concentrations (5%) was similar in the wild-type and in the tpk2 null mutant strains suggesting that under these conditions germination was triggered through a PKA-independent pathway.