No abstract

The serine/threonine kinase Akt (also called protein kinase B) is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. Here, we identify an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 was replaced with alanine, formed abnormal elongated shapes and exhibited limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility.

GDNF signaling through the Ret receptor tyrosine kinase is critical for ureteric bud branching morphogenesis during kidney development, yet few of the downstream genes are currently known. We find that the ETS transcription factors Etv4 and Etv5 are positively regulated by Ret signaling in the ureteric bud tips. Etv4−/−, Etv5+/− mice display either renal agenesis or severe hypodysplasia, while kidney development fails completely in double homozygotes. We identify several genes whose expression in the ureteric bud depends on Etv4 and Etv5, including Cxcr4, Myb, Met, Mmp14. Thus, Etv4 and Etv5 are key components of a gene network downstream of Ret that promotes and controls renal branching morphogenesis.

The serine/threonine protein kinase Akt is involved in a variety of cellular processes including cell proliferation, survival, metabolism and gene expression. It is essential in vascular endothelial growth factor (VEGF)-mediated angiogenesis; however, it is not known how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching and the formation of networks during angiogenesis. Here we report that Akt-mediated phosphorylation of Girdin, an actin-binding protein, promotes VEGF-dependent migration of endothelial cells and tube formation by these cells. We found that exogenously delivered adenovirus harbouring Girdin short interfering RNA in Matrigel embedded in mice, markedly inhibited VEGF-mediated angiogenesis. Targeted disruption of the Girdin gene in mice impaired vessel remodelling in the retina and angiogenesis from aortic rings, whereas Girdin was dispensable for embryonic vasculogenesis. These findings demonstrate that the Akt/Girdin signalling pathway is essential in VEGF-mediated postneonatal angiogenesis.

The c-ret proto-oncogene encodes a transmembrane tyrosine kinase that contains a cadherin-like structure in the extracellular domain (9,10,19,22,23). Its expression was detected at high levels in the peripheral nervous systems such as the enteric and autonomic nervous systems as well as in the excretory system during embryogenesis (1a, 15, 25). In addition, it is expressed preferentially in human tumors such as neuroblastoma, pheochromocytoma, and thyroid medullary carcinoma (8,18,24). Since the peripheral nervous systems and tumors mentioned above derive from neural crest cells, the physiological function of the c-ret proto-oncogene appears related to their normal growth and differentiation.Recent studies revealed that germ line mutations in the c-ret proto-oncogene are associated with the development of four different neural crest disorders (neurocristopathies): multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma, and Hirschsprung's disease (2,4,5,7,13,16). MEN 2A and MEN 2B are autosomal dominant cancer syndromes characterized by the development of medullary thyroid carcinoma and pheochromocytoma. MEN 2B is distinguished from MEN 2A by a more complex phenotype including mucosal neuroma, hyperganglionosis of the gastrointestinal tract, and marfanoid habitus. MEN 2A and familial medullary thyroid carcinoma mutations always involve cysteine residues present in the extracellular domain of the c-ret proto-oncogene (4, 12, 13). These cysteine residues are conserved in both human and mouse c-ret proto-oncogenes, suggesting that they are important for normal conformation of the c-Ret protein (9,22,23). On the other hand, a single point mutation in exon 16 of the tyrosine kinase domain has been found in 95% of patients with MEN 2B (6). This difference of the mutation sites may account for different phenotypes of MEN 2A and MEN 2B. Alternatively, it is possible that the diverse phenotypes observed in MEN 2A and MEN 2B are due to mutations in other modifier genes.Hirschsprung's disease is a developmental disorder of the enteric nervous system, inherited in an autosomal dominant manner with incomplete penetrance and variant expressivity. Several mutations have been found in different domains of the c-ret proto-oncogene, including the extracellular and tyrosine kinase domains (5, 16). Since mice homozygous for c-ret disruption showed phenotypes similar to Hirschsprung's disease (20), it is likely that the abnormalities observed in Hirschsprung's disease are caused by inactivation of the c-Ret function. On the other hand, MEN 2A and MEN 2B mutations might represent gain-of-function mutations.To elucidate the mechanism of development of MEN 2A syndrome, we introduced MEN 2A mutations in the extracellular domain of the c-ret proto-oncogene and analyzed their functions. Biochemical analysis of the Ret protein with MEN 2A mutations indicated that it is activated by ligand-independent dimerization on the cell surface. In addition, we showed that a mutation in a putative Ca 2ϩ -binding site of the ...