Necl-4 enhances the PLCγ-c-Raf-MEK-ERK pathway without affecting internalization of VEGFR2.

We have reported that knockdown of Necl-4 decreases vascular endothelial growth factor (VEGF)-induced phosphorylation of extracellular signal-regulated kinase (ERK) without affecting phosphorylation of VEGF receptor 2 (VEGFR2) in sparsely cultured human umbilical vein endothelial cells (HUVECs). However, the underlying molecular mechanism is unknown. Compared with control HUVECs, VEGF-induced phosphorylation of phospholipase Cγ (PLCγ), c-Raf, mitogen-activated protein kinase/ERK kinase (MEK) and ERK were all inhibited in Necl-4-knockdown HUVECs. However, VEGF-induced internalization of VEGFR2 was not different between control and Necl-4-knockdown HUVECs. We have reported that protein-tyrosine phosphatase, non-receptor type 13 (PTPN13) and Rho-associated kinase (ROCK) are involved in the Necl-4-knockdown-induced inhibition of the VEGF-induced activation of Rac1. However, the effects of Necl-4-knockdown on VEGF-induced phosphorylation of VEGFR2 and ERK were not affected either by knockdown of PTPN13 or by ROCK inhibitors. These results suggest that Necl-4 enhances VEGF-induced activation of PLCγ-c-Raf-MEK-ERK pathway without affecting the phosphorylation and internalization of VEGFR2.

Thermotolerant genes essential for survival at a critical high temperature in thermotolerant ethanologenic Zymomonas mobilis TISTR 548.

BACKGROUND: High-temperature fermentation (HTF) technology is expected to reduce the cost of bioconversion of biomass to fuels or chemicals. For stable HTF, the development of a thermotolerant microbe is indispensable. Elucidation of the molecular mechanism of thermotolerance would enable the thermal stability of microbes to be improved. RESULTS: Thermotolerant genes that are essential for survival at a critical high temperature (CHT) were identified via transposon mutagenesis in ethanologenic, thermotolerant Zymomonas mobilis TISTR 548. Surprisingly, no genes for general heat shock proteins except for degP were included. Cells with transposon insertion in these genes showed a defect in growth at around 39 °C but grew normally at 30 °C. Of those, more than 60% were found to be sensitive to ethanol at 30 °C, indicating that the mechanism of thermotolerance partially overlaps with that of ethanol tolerance in the organism. Products of these genes were classified into nine categories of metabolism, membrane stabilization, transporter, DNA repair, tRNA modification, protein quality control, translation control, cell division, and transcriptional regulation. CONCLUSIONS: The thermotolerant genes of Escherichia coli and Acetobacter tropicalis that had been identified can be functionally classified into 9 categories according to the classification of those of Z. mobilis, and the ratio of thermotolerant genes to total genomic genes in Z. mobilis is nearly the same as that in E. coli, though the ratio in A. tropicalis is relatively low. There are 7 conserved thermotolerant genes that are shared by these three or two microbes. These findings suggest that Z. mobilis possesses molecular mechanisms for its survival at a CHT that are similar to those in E. coli and A. tropicalis. The mechanisms may mainly contribute to membrane stabilization, protection and repair of damage of macromolecules and maintenance of cellular metabolism at a CHT. Notably, the contribution of heat shock proteins to such survival seems to be very low.

The Cell Adhesion Molecule Necl-4/CADM4 Serves as a Novel Regulator for Contact Inhibition of Cell Movement and Proliferation.

Contact inhibition of cell movement and proliferation is critical for proper organogenesis and tissue remodeling. We show here a novel regulatory mechanism for this contact inhibition using cultured vascular endothelial cells. When the cells were confluently cultured, Necl-4 was up-regulated and localized at cell-cell contact sites where it cis-interacted with the vascular endothelial growth factor (VEGF) receptor. This interaction inhibited the tyrosine-phosphorylation of the VEGF receptor through protein-tyrosine phosphatase, non-receptor type 13 (PTPN13), eventually reducing cell movement and proliferation. When the cells were sparsely cultured, Necl-4 was down-regulated but accumulated at leading edges where it inhibited the activation of Rho-associated protein kinase through PTPN13, eventually facilitating the VEGF-induced activation of Rac1 and enhancing cell movement. Necl-4 further facilitated the activation of extracellular signal-regulated kinase 1/2, eventually enhancing cell proliferation. Thus, Necl-4 serves as a novel regulator for contact inhibition of cell movement and proliferation cooperatively with the VEGF receptor and PTPN13.