SUMMARY Both pro- and anti-oncogenic properties have been attributed to EphA2 kinase. We report that a possible cause for this apparent paradox is diametrically opposite roles of EphA2 in regulating cell migration and invasion. While activation of EphA2 with its ligand ephrin-A1 inhibited chemotactic migration of glioma and prostate cancer cells, EphA2 overexpression promoted migration in a ligand-independent manner. Surprisingly, the latter effects required phosphorylation of EphA2 on serine 897 by Akt, and S897A mutation abolished ligand-independent promotion of cell motility. Ephrin-A1 stimulation of EphA2 negated Akt activation by growth factors and caused EphA2 dephosphorylation on S897. In human astrocytoma, S897 phosphorylation was correlated with tumor grades and Akt activation, suggesting that the Akt-EphA2 crosstalk may contribute to brain tumor progression.

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.

Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis

Histone (de)acetylation is important for the regulation of fundamental biological processes such as gene expression and DNA recombination. Distinct classes of histone deacetylases (HDACs) have been identified, but how they are regulated in vivo remains largely unexplored. Here we describe results demonstrating that HDAC4, a member of class II human HDACs, is localized in the cytoplasm and/or the nucleus. Moreover, we have found that HDAC4 interacts with the 14-3-3 family of proteins that are known to bind specifically to conserved phosphoserine-containing motifs. Deletion analyses suggested that S246, S467, and S632 of HDAC4 mediate this interaction. Consistent with this, alanine substitutions of these serine residues abrogated 14-3-3 binding. Although these substitutions had minimal effects on the deacetylase activity of HDAC4, they stimulated its nuclear localization and thus led to enhanced transcriptional repression. These results indicate that 14-3-3 proteins negatively regulate HDAC4 by preventing its nuclear localization and thereby uncover a novel regulatory mechanism for HDACs.Specific lysine acetylation of histones and nonhistone proteins has been recently recognized as a major mechanism by which eukaryotic transcription is regulated (12,23,24,44,45,56,57). Such acetylation is reversible and dynamic in vivo, and its level is governed by the opposing actions of histone acetyltransferases and histone deacetylases (HDACs). Distinct classes of HDACs have been identified in mammals (21, 36). Class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) are homologous to yeast Rpd3 (8,16,49,60,61). HDAC1 and HDAC2 interact with each other and form the catalytic core of Sin3 and NuRD complexes, both of which play important roles in transcriptional repression and gene silencing (26,51,53,54,58,(63)(64)(65). Various transcriptional repressors recruit these complexes to inhibit transcription (reviewed in references 15, 45, and 56). Class II HDACs (HDAC4, HDAC5, HDAC6, and HDAC7) contain domains significantly similar to the catalytic domain of yeast Hda1 (9,11,20,33,41,52,55). HDAC4, HDAC5, and HDAC7 are homologous, whereas HDAC6 has two Hda1-related catalytic domains and a unique Cys-and Hisrich C-terminal domain. HDAC4 and HDAC5 interact with the MEF2 transcription factors (28,33,55), and this interaction is regulated (30, 62). Related to this, MITR/HDRP, a protein related to the N-terminal part of HDAC4, HDAC5, and HDAC7, binds to MEF2s and represses transcription (43,66). Moreover, HDAC4, HDAC5, and HDAC7 were found to interact with the nuclear receptor corepressors 17,20). These new findings suggest that like class I HDACs, some class II HDACs are recruited to promoters to inhibit transcription. One interesting but unaddressed question is how the function of HDACs is regulated in vivo.While HDAC1, HDAC2, and HDAC3 are nuclear, the plant deacetylase HD2 is a nucleolar protein (8, 31). Miska et al. reported that the HDAC4 protein lacking the N-terminal 117 residues is cytoplasmic or nuclear in HeLa cells (33), whereas Fischl...

The essentially infinite expansion potential and pluripotency of human embryonic stem cells (hESCs) makes them attractive for cell-based therapeutics. In contrast to mouse embryonic stem cells (mESCs), hESCs normally undergo high rates of spontaneous apoptosis and differentiation, making them difficult to maintain in culture. Here we demonstrate that p53 protein accumulates in apoptotic hESCs induced by agents that damage DNA. However, despite the accumulation of p53, it nevertheless fails to activate the transcription of its target genes. This inability of p53 to activate its target genes has not been observed in other cell types, including mESCs. We further demonstrate that p53 induces apoptosis of hESCs through a mitochondrial pathway. Reducing p53 expression in hESCs in turn reduces both DNA damage-induced apoptosis as well as spontaneous apoptosis. Reducing p53 expression also reduces spontaneous differentiation and slows the differentiation rate of hESCs. Our studies reveal the important roles of p53 as a critical mediator of human embryonic stem cells survival and differentiation.Human embryonic stem cells (hESCs) 3 are capable of essentially unlimited self-renewal and retain the developmental potential to differentiate into almost any cell type. These characteristics of hESCs make them attractive for tissue and cellbased therapies (1, 2). Previously, basic fibroblast growth factor and activin A were identified as self-renewal factors (3-6). However, for reasons that are not clear, hESCs often display high rates of spontaneous apoptosis and differentiation in culture, thus making the process of expanding these cells highly inefficient (3, 7-10). For example, Dravid et al. (8) reported that, under routine culture conditions, Ͼ30% of hESCs undergo spontaneous apoptosis. Furthermore, Ezashi et al. (12) showed that nearly 40% of hESCs undergo spontaneous differentiation after 12 days of culture in normoxic conditions. Finally, Maitra et al. (13) reported that multiple passages of hESCs can cause genomic alterations, which may limit the therapeutic application of hESCs. In contrast to hESCs, mouse embryonic stem cells (mESCs) undergo lower rates of spontaneous apoptosis and differentiation (14). Moreover, they maintain their pluripotency and genomic stability longer than hESCs (15). The reason for these different species-specific phenotypes in embryonic stem cells is currently unknown.The p53 tumor suppressor gene is a strong candidate for playing a role in the observed phenotypes of hESCs, because it regulates various cellular processes, including apoptosis, differentiation, and genomic integrity (16). In many cell types p53 plays a crucial role in controlling apoptosis and cell cycle arrest when these cells are exposed to stress-inducing conditions (17). In response to stress, p53 accumulates and transactivates downstream target genes such as mdm2 (responsible for the feedback degradation circuitry of p53), p21 (responsible for cell cycle control), bax, noxa, and puma (responsible for DNA damage-induced ...