Evan Ingley

High affinity binding of inositol phosphates and phosphoinositides to the pleckstrin homology domain of RAC/protein kinase B and their influence on kinase activity

The influence of inositol phosphates and phosphoinositides on the α isoform of the RAC-protein kinase B (RAC/PKB) was studied using purified wild type and mutant kinase preparations and a recombinant pleckstrin homology (PH) domain. Binding of inositol phosphates and phosphoinositides to the PH domain was measured as the quenching of intrinsic tryptophan fluorescence. Inositol phosphates and D3-phosphorylated phosphoinositides bound with affinities of 1-10 μM and 0.5 μM, respectively. Similar values were obtained using RAC/PKB expressed and purified from baculovirus-infected Sf9 cells in the fluorescence assay. The influence of synthetic dioctanoyl derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate on the activity of RAC/PKB purified from transfected COS-1 cells was studied. Phosphatidylinositol 3,4,5-trisphosphate was found to inhibit the RAC/PKB kinase activity with half-maximal inhibition at 2.5 μM. In contrast, phosphatidylinositol 3,4-bisphosphate stimulated kinase activity (half-maximal stimulation at 2.5 μM). A mutant RAC/PKB protein lacking the PH domain was not affected by D3-phosphorylated phosphoinositides. These results demonstrate that the PH domain of RAC/PKB binds inositol phosphates and phosphoinositides with high affinity, and suggest that the products of the phosphatidylinositide 3-kinase can act as both a membrane anchor and modulator of RAC/PKB activity. The data also provide further evidence for a link between phosphatidylinositide 3-kinase and RAC/PKB regulation.

Lyn tyrosine kinase is essential for erythropoietin- induced differentiation of J2E erythroid cells

Erythropoietin stimulates the immature erythroid J2E cell line to terminally differentiate and maintains the viability of the cells in the absence of serum. In contrast, a mutant J2E clone (J2E‐NR) fails to mature in response to erythropoietin; however, it remains viable in the presence of the hormone. We have shown previously that intracellular signalling is disrupted in the J2E‐NR cell line and that tyrosine phosphorylation is dramatically reduced after erythropoietin stimulation. In this study we investigated the defect in J2E‐NR cells that is responsible for their inability to differentiate. Screening of numerous signalling molecules revealed that the lyn tyrosine kinase appeared to be absent from J2E‐NR cells. On closer examination, both lyn mRNA and protein content were reduced >500‐fold. Consistent with a defect in lyn, amphotropic retroviral infection of J2E‐NR cells with lyn restored the ability of the cells to synthesize haemoglobin and enabled the cells to mature morphologically. Conversely, the ability of J2E cells to differentiate in response to epo was severely curtailed when antisense lyn oligonucleotides or a dominant negative lyn were introduced into the cells. However, erythropoietin‐supported viability was unaffected by reducing lyn activity. The ability of two other erythropoietin‐responsive cell lines (R11 and R24) to differentiate in response to the hormone was also reduced by dominant negative lyn. Finally, co‐immunoprecipitation and yeast two‐hybrid analyses indicated that lyn directly associated with the erythropoietin receptor complex. These data indicate for the first time an essential role for lyn in erythropoietin‐initiated differentiation of J2E cells but not in the maintenance of cell viability.

The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and Hop is located in the dimerization domain of hsp90

Structurally related tetratricopeptide repeat motifs in steroid receptor-associated immunophilins and the STI1 homolog, Hop, mediate the interaction with a common cellular target, hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA library. All isolated clones encoded the intact carboxyl terminus of hsp90 and overlapped with a common region corresponding to amino acids 558–724 of murine hsp84. The interaction was confirmed in vitro with bacterially expressed CyP40 and deletion mutants of hsp90β and was delineated further to a 124-residue COOH-terminal segment of hsp90. Deletion of the conserved MEEVD sequence at the extreme carboxyl terminus of hsp90 precludes interaction with CyP40, signifying an important role for this motif in hsp90 function. We show that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90 COOH-terminal region. Our results are consistent with a common tetratricopeptide repeat interaction site for Hop and steroid receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.

A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61β subunit

We report here the isolation of a new member of the ADP‐ribosylation factor (ARF)‐like family (ARL‐6) present in the J2E erythroleukemic cell line, but not its myeloid variants. Consistent with this lineage‐restricted expression, ARL‐6 mRNA increased with erythropoietin‐induced maturation of J2E cells, and decreased with interleukin 6‐induced differentiation of M1 monoblastoid cells. In tissues, ARL‐6 mRNA was most abundant in brain and kidney. While ARL‐6 protein was predominantly cytosolic, its membrane association increased following exposure to GTP‐γS, like many members of the ARF/ARL family. Using the yeast two‐hybrid system, six molecules which interact with ARL‐6 were identified including SEC61β, a subunit of the heterotrimeric protein conducting channel SEC61p. Co‐immunoprecipitation of ARL‐6 confirmed a stable association between ARL‐6 and SEC61β in COS cells. These results demonstrate that ARL‐6, a novel member of the ADP‐ribosylation factor‐like family, interacts with the SEC61β subunit.

Functions of the Lyn tyrosine kinase in health and disease

Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn−/− mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up) have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and B-cell lymphocytic leukaemia (BCLL). Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer.Lay AbstractTo relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information input, Lyn can positively or negatively regulate the information output. This ability of Lyn to be able to both turn on and turn off the relay of information inside cells makes it difficult to fully understand its precise function in each specific circumstance. Lyn has important functions for cells involved in blood development, including different while blood cells as well as red blood cells, and in particular for the immune cells that produce antibodies (B-cells), as exemplified by the major B-cell abnormalities that mice with mutations in the Lyn gene display. Certain types of leukaemia and lymphoma appear to have too much Lyn activity that in part causes the characteristics of these diseases, suggesting it may be a good target to develop new anti-leukaemia drugs. Furthermore, some specific types, and even specific subtypes, of solid cancers, e.g. prostate, brain and breast cancer can also have abnormal regulation of Lyn. Consequently, targeting this protein in these cancers could also prove to be beneficial.

HLS7, a hemopoietic lineage switch gene homologous to the leukemia‐inducing gene MLF1

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid‐specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin‐ or chemically‐induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin‐responsive colonies in semi‐solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

New insights into the regulation of erythroid cells

The regulation of erythroid cells is complex and occurs at multiple levels. Erythroid precursors, once committed to this lineage, develop in association with specific macrophages within erythroblastic islands. While erythropoietin (Epo) is the principal regulator of erythroid progenitors, other cytokines and nuclear hormones also play an important role in the maturation of these cells. Signalling from the Epo‐receptor activates several pathways, including the JAK/STAT, ras/raf/MAP kinase and PI3 kinase/Akt cascades to promote cell survival, proliferation and differentiation. Transcription factors such as GATA‐1, EKLF and NF‐E2 are crucial for progression along the erythroid maturation pathway; these, and a myriad of other transcription factors, must be expressed at the correct developmental stage for normal red blood cells to be formed. IUBMB Life, 56: 177‐184, 2004

MADM, a Novel Adaptor Protein That Mediates Phosphorylation of the 14-3-3 Binding Site of Myeloid Leukemia Factor 1

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3ζ and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3ζ via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3ζ, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

Crystal structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound state

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its “active” conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.

Csk-binding protein mediates sequential enzymatic down-regulation and degradation of Lyn in erythropoietin-stimulated cells.

We have shown previously that the Src family kinase Lyn is involved in differentiation signals emanating from activated erythropoietin (Epo) receptors. The importance of Lyn to red cell maturation has been highlighted by Lyn-/- mice developing anemia. Here we show that Lyn interacts with C-terminal Src kinase-binding protein (Cbp), an adaptor protein that recruits negative regulators C-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk). Lyn phosphorylated Cbp on several tyrosine residues, including Tyr314, which recruited Csk/Ctk to suppress Lyn kinase activity. Intriguingly, phosphorylated Tyr314 also bound suppressor of cytokine signaling 1 (SOCS1), another well characterized negative regulator of cell signaling, resulting in elevated ubiquitination, and degradation of Lyn. In Epo-responsive primary cells and cell lines, Lyn rapidly phosphorylated Cbp, suppressing Lyn kinase activity via Csk/Ctk within minutes of Epo stimulation; hours later, SOCS1 bound to Cbp and was involved in the ubiquitination and turnover of Lyn protein. Thus, a single phosphotyrosine residue on Cbp coordinates a two-phase process involving distinct negative regulatory pathways to inactivate, then degrade, Lyn.