Mutations in the human endoglin gene result in hereditary hemorrhagic telangiectasia type 1, a vascular disorder characterized by multisystemic vascular dysplasia, arteriovenous malformations, and focal dilatation of postcapillary venules. Previous studies have implicated endoglin in the inhibition of cell migration in vivo and in vitro. In the course of studies to address the relationship of the conserved cytosolic domain to endoglin function, we identified zyxin, a LIM domain protein that is concentrated at focal adhesions, as an interactor with endoglin in human umbilical vein vascular endothelial cells. This interaction is localized within the 47-amino acid carboxyl-terminal cytosolic domain of endoglin, and maps within zyxin residues 326 -572. The endoglin-zyxin interaction was found to be largely mediated by the third LIM domain of zyxin, and is specific for endoglin because the homologous cytosolic domain of the transforming growth factor-␤ type III receptor, betaglycan, fails to interact with zyxin. Expression of endoglin is associated with reduction of zyxin, as well as its interacting proteins p130 cas and CrkII, from a focal adhesion protein fraction, and this reduction is correlated with inhibition of cell migration. We also show that endoglin-dependent: (i) inhibition of cell migration, (ii) reduction of focal adhesion-associated p130 cas /CrkII protein levels, (iii) tyrosine phosphorylation of p130 cas , and (iv) focal adhesion-associated endoglin levels are mediated by the cytosolic domain of endoglin. These results suggest a novel mechanism of endoglin function involving its interaction with LIM domain-containing proteins, and associated adapter proteins, affecting sites of focal adhesion.Endoglin is a 190-kDa homodimeric transmembrane glycoprotein composed of 95-kDa disulfide-linked subunits (1). Mutations in the gene encoding endoglin have been linked to the human disease: hereditary hemorrhagic telangiectasia type 1 (HHT1) 1 (2, 3), an autosomal dominant inherited vascular disorder characterized by localized vascular dysplasia and a tendency toward arteriovenous malformations. Arteriovenous malformations occur in ϳ20% of patients and are associated with life-threatening complications including stroke and brain abscess. Pathological features found in HHT telangiectases include focal dilatation of postcapillary venules and a prominence of actin stress fibers in pericytes (4 -6). Mice lacking endoglin die from defective angiogenesis characterized by failure of vascular smooth muscle investment of embryonic blood vessels, suggesting a defect in vascular smooth muscle cell development (7). The molecular mechanism underlying the angiogenic phenotype in the endoglin null mouse remains unclear.In vivo data points to a role for endoglin in the vascular response to injury. Findings from this laboratory showed that endoglin was expressed in human aortic smooth muscle cells in atherosclerotic plaques but was absent from normal smooth muscle (8). Ma et al. (9) found that expression of endoglin in normal p...

The transcription factor C/EBP␣ is a critical mediator of myeloid differentiation and is often functionally impaired in acute myeloid leukemia. Recent studies have suggested that oncogenic FLT3 activity disrupts wild-type C/EBP␣ function via phosphorylation on serine 21 (S21). Despite the apparent role of pS21 as a negative regulator of C/EBP␣ transcription activity, the mechanism by which phosphorylation tips the balance between transcriptionally competent and inhibited forms remains unresolved. In the present study, we used immuno-affinity purification combined with quantitative mass spectrometry to delineate the proteins associated with C/EBP␣ on chromatin. We identified DEK, a protein with genetic links to leukemia, as a member of the C/EBP␣ complexes, and demonstrate that this association is disrupted by S21 phosphorylation. We confirmed that DEK is recruited specifically to chromatin with C/EBP␣ to enhance GCSFR3 promoter activation. In addition, we demonstrated that genetic depletion of DEK reduces the

Determination of the functional relationship between the transforming growth factor-␤ (TGF␤) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGF␤1 caused recruitment of ALK1 into a complex with endoglin in human umbilical vein endothelial cells (HUVECs). Therefore, we examined TGF␤ receptor-dependent phosphorylation of endoglin by the constitutively active forms of the TGF␤ type I receptors ALK1, ALK5, and the TGF␤ type II receptor, T␤RII. Of these receptors, T␤RII preferentially phosphorylated endoglin on cytosolic domain serine residues Ser 634 and Ser 635. Removal of the carboxyl-terminal tripeptide of endoglin, which comprises a putative PDZ-liganding motif, dramatically increased endoglin serine phosphorylation by all three receptors, suggesting that the PDZ-liganding motif is important for the regulation of endoglin phosphorylation. Constitutively active (ca)ALK1, but not caALK5, phosphorylated endoglin on cytosolic domain threonine residues. caALK1-mediated threonine phosphorylation required prior serine phosphorylation, suggesting a sequential mechanism of endoglin phosphorylation. Wild-type, but not a threonine phosphorylation-defective endoglin mutant blocked cell detachment and the antiproliferative effects of caALK1 expressed in HUVECs. These results suggest that ALK1 is a preferred TGF␤ receptor kinase for endoglin threonine phosphorylation in HUVECs and indicate a role for endoglin phosphorylation in the regulation of endothelial cell adhesion and growth by ALK1.In humans, there are seven type I and five type II TGF␤ 3 receptors currently known (1). TGF␤ type I/II receptors are serine and threonine (S/T) kinases, although the significance of the serine-versus threonine-phosphorylating activities of the TGF␤ receptors is not clear. The TGF␤ type I receptors include activin-like kinase 1 (ALK1) and T␤RI, also known as ALK5. ALK1 and ALK5 associate with the type II TGF␤ receptor, T␤RII (2). The binding of TGF␤ ligands by T␤RII induces the type I receptor to associate with T␤RII, allowing the latter to phosphorylate the type I receptor and activate its kinase domain (3). The activated type I receptor propagates signaling by phosphorylating the Smad family of transcription corepressors and coactivators (4). Endoglin is a type III TGF␤ receptor (5, 6) that is also a substrate for T␤RII-and ALK5-catalyzed phosphorylation (7), although the relevance of endoglin phosphorylation and the ability of endoglin to serve as a substrate for ALK1 have not been demonstrated.Phosphorylation of human endoglin occurs in endothelial cells and mouse fibroblasts (8,9). These studies demonstrate that endoglin is phosphorylated on serine (9) and threonine residues (10). T␤RII and ALK5 interact with endoglin, and, as a result of these associations, endoglin is phosphorylated on its cytosolic domain (CD) (7). No previous studies have identified the specific endoglin residues that were phosphorylated, examined the kinase specificities of...

Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1beta (SH2B1beta), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e.g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation.