Rositsa I. Koleva

Endoglin structure and function - Determinants of endoglin phosphorylation by transforming growth factor-beta receptors

Determination of the functional relationship between the transforming growth factor-β (TGFβ) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGFβ1 caused recruitment of ALK1 into a complex with endoglin in human umbilical vein endothelial cells (HUVECs). Therefore, we examined TGFβ receptor-dependent phosphorylation of endoglin by the constitutively active forms of the TGFβ type I receptors ALK1, ALK5, and the TGFβ type II receptor, TβRII. Of these receptors, TβRII preferentially phosphorylated endoglin on cytosolic domain serine residues Ser634 and Ser635. 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 TGFβ 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.

Endoglin structure and function: Determinants of endoglin phosphorylation by TGFβ receptors

Determination of the functional relationship between the transforming growth factor-β (TGFβ) receptor proteins endoglin and ALK1 is essential to the understanding of the human vascular disease, hereditary hemorrhagic telangiectasia. TGFβ1 caused recruitment of ALK1 into a complex with endoglin in human umbilical vein endothelial cells (HUVECs). Therefore, we examined TGFβ receptor-dependent phosphorylation of endoglin by the constitutively active forms of the TGFβ type I receptors ALK1, ALK5, and the TGFβ type II receptor, TβRII. Of these receptors, TβRII preferentially phosphorylated endoglin on cytosolic domain serine residues Ser634 and Ser635. 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 TGFβ 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.

Regulation of Jak2 function by phosphorylation of Tyr317 and Tyr637 during cytokine signaling.

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

C/EBPα and DEK coordinately regulate myeloid differentiation

The transcription factor C/EBPα 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/EBPα function via phosphorylation on serine 21 (S21). Despite the apparent role of pS21 as a negative regulator of C/EBPα 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/EBPα on chromatin. We identified DEK, a protein with genetic links to leukemia, as a member of the C/EBPα complexes, and demonstrate that this association is disrupted by S21 phosphorylation. We confirmed that DEK is recruited specifically to chromatin with C/EBPα to enhance GCSFR3 promoter activation. In addition, we demonstrated that genetic depletion of DEK reduces the ability of C/EBPα to drive the expression of granulocytic target genes in vitro and disrupts G-CSF-mediated granulocytic differentiation of fresh human BM-derived CD34(+) cells. Our data suggest that C/EBPα and DEK coordinately activate myeloid gene expression and that S21 phosphorylation on wild-type C/EBPα mediates protein interactions that regulate the differentiation capacity of hematopoietic progenitors.

Tyrosines 868, 966, and 972 in the Kinase Domain of JAK2 Are Autophosphorylated and Required for Maximal JAK2 Kinase Activity

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.

Abstract B51: Dynamic remodeling of CEBPα complexes in myeloid differentiation

Acute myeloid leukemia (AML) remains a highly lethal malignancy with limited therapeutic options. An integral component of the AML phenotype is an inability of hematopoietic progenitors to differentiate into mature myeloid cells. Mounting evidence indicates that the basic leucine zipper transcription factor C/EBPα plays a critical role in development of granulocytes, and that impaired function of C/EBPα may disrupt myeloid differentiation and ultimately lead to blast crisis in AML. Recent studies demonstrated that oncogenic Flt3 signaling leads to phosphorylation of C/EBPα on serine 21, with concomitant impairment of myeloid differentiation in cell line models and primary hematopoietic cells. While these data provide compelling evidence for the central role of C/EBPα in normal hematopoiesis and leukemia, the mechanism by which pS21 disrupts differentiation remains unresolved. Given the general thesis that cellular control of biological processes is orchestrated through a delicate balance of protein‐protein interactions and post‐translational modifications, we speculated that C/EBPα phosphorylation catalyzes the assembly of a transcriptionally inactive protein complex. To test this hypothesis we established affinity tagged C/EBPα under control of a tet‐inducible promoter in myeloid progenitors with constitutive Flt3 activity. Treatment of these cells with Flt3 inhibitors modulated pS21 in a dose‐dependent manner. Next advanced quantitative proteomics methodology, including true nanoflow LC coupled with multidimensional fractionation and on‐column iTRAQ stable isotope labeling, was used to monitor remodeling of C/EBPα protein complexes as a function of pS21. Our data represent by far the largest catalog C/EBPα interactors involved in chromatin organization, transcriptional modulation, and cell cycle regulation. Furthermore, our quantitative proteomics data demonstrate that 1) C/EBPα interacts with proteins genetically linked to leukemia (DEK, SET and other); and 2) many of these interact with C/EBPα in a phosphorylation‐dependent manner. Our functional validation studies show that DEK and SET bind to C/EBPα‐target promoters in C/EBPα‐dependent manner to enhance C/EBPα attachment to its DNA binding site. Knock‐down of newly‐identified, leukemia‐associated interactors, DEK and SET, reduce the ability of C/EBPα to drive expression of granulocytic target genes. Furthermore the depletion of DEK and SET reduce capacity of myeloid cells to undergo terminal granulocytic differentiation. Our data demonstrate that phosphorylation on serine 21 modulates association of C/EBPα with protein partners that are functionally relevant for myeloid differentiation. Our ability to quantitatively monitor multiple leukemia‐related gene products in the context of C/EBPα protein complexes provides valuable insight into the mechanisms by which oncogenic kinase activity disrupts transcription and leads to leukemogenesis. Citation Information: Cancer Res 2009;69(23 Suppl):B51.