In an increasing number of hematopoietic cytokine receptor HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activa-erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase tion. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F) were expressed in HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late ery-SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglo-throid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in binization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO may enforce mitogenesis. ᭧
We readily produced recombinant pro-macrophage stimulating protein in a mammalian expression system, but it was only weakly active after proteolytic activation. Active macrophage stimulating protein is a disulfide-bonded heterodimer, but in our hands, the subunits of recombinant macrophage stimulating protein were mostly not disulfide bonded. 3 Ala macrophage stimulating protein were fully disulfide linked, and the mutant macrophage stimulating protein had 10 -20-fold higher specific activity than the wild type recombinant macrophage stimulating protein.Macrophage-stimulating protein (MSP) 1 was originally purified from human serum as a protein that enhances the chemotactic response of murine peritoneal macrophages to the C5a fraction of complement (1). In a screen for proteins that contain kringle domains, Degen and co-workers (2) identified a cDNA that was most closely related to hepatocyte growth factor (HGF) and hence named it HGF-like (HGF-L). MSP and HGF-L were later shown to be the same molecule (3). MSP and HGF (4) are plasminogen-related growth factors and appear to exert their biological effects as ligands for the receptor protein tyrosine kinases RON (5, 6) and MET (7), respectively. The murine orthologue of human RON is also known as STK (8, 9). Like plasminogen, these growth factors require proteolytic activation for biological activity, which occurs between homologous Arg-Val residues and generates heterodimeric disulfidebonded subunits (10). The ␣ subunit contains four kringle domains, whereas the  subunit contains an inactive serine protease domain (SPD). It is not established which protease is responsible for the in vivo activation of MSP, but several proteases, such as human plasma kallikrein, are reported to efficiently activate MSP in vitro (10). The biological role of the RON/MSP cognate receptor/ligand pair is still unclear. Experimental strategies to address this issue include analysis of the expression pattern of RON in situ, 2 and a survey of the effects of MSP in a variety of in vitro bioassays. Because the latter requires a steady supply of MSP, we began producing recombinant MSP. Our initial attempts to produce recombinant MSP generated preparations with a dramatically reduced specific activity (ϳ50-fold) as compared with material that was purified from a nonrecombinant source. The following studies were undertaken in attempts to determine the underlying mechanism for the reduced activity and to investigate how the activity might be restored to levels observed with naturally produced protein. (11)) was a gift of Professor Sandra Degen, University of Cincinnati. The Cys 672 3 Ala MSP mutant was constructed by polymerase chain reaction from the MSP cDNA with a mutant primer containing a change from TG to GC at nucleotides 2024 and 2025 (5Ј-CACAACGCCTGGGTCCTGGAAG-3Ј) and a downstream primer (5Ј-CTGGCAACTAGAAGGCACAGTCG-3Ј) complementary to vector pCDNA3 (Invitrogen, San Diego, CA). All contructs were verified by DNA sequencing. MATERIALS AND METHODS Construction of MSP cDNAs-The human MS...
In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F ) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.
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