Hematopoietic cell growth, di erentiation, and commitment to a restricted lineage are guided by a set of cytokines acting exclusively on cells expressing the corresponding cytokine receptor. The macrophage colony stimulating factor (M-CSF, also termed CSF-1) and its cognate receptor, the tyrosine kinase c-Fms, are essential for monocyte and macrophage development. The underlying molecular mechanism, however, is poorly understood. Here we identi®ed a novel Fms-interacting protein (FMIP, MW 78 kDa) which binds transiently via its Nterminal 144 residues to the cytoplasmic domain of activated Fms-molecules. Binding of FMIP was paralleled by rapid tyrosine phosphorylation within the binding domain which drastically reduced its ability to associate with Fms. Binding was speci®c as evidenced by co-immunoprecipitation and association with recombinant GST-Fms fusion proteins. No binding was observed with the tyrosine phosphorylated cytoplasmic domains of c-Kit, TrkA, c-Met, and the insulin receptor. The role of FMIP in hematopoietic di erentiation was studied in the bipotential myeloid progenitor cell line, FDC-P1Mac11. Overexpression of FMIP prevented M-CSF induced macrophage di erentiation. Instead, cells di erentiated into granulocytes. Our data suggest that the level of FMIP expression could form a threshold that decides about di erentiation either into macrophages or into granulocytes.
Small heat shock proteins (sHsps) exist in dynamic oligomeric complexes and display diverse biological functions ranging from chaperone properties to modulator of apoptosis. So far, the role of stress-dependent phosphorylation of mammalian sHsps for its structure and function has been analyzed by using various phosphorylation site mutants overexpressed in different cell types as well as by non-exclusive inhibitors of the p38 MAPK cascade. Here we investigate the role of phosphorylation of endogenous sHsp in a genetic model lacking the major Hsp25 kinase, the MAP kinase-activated protein kinase MK2. We demonstrate that in MK2-deficient fibroblasts, where no stress-dependent phosphorylation of Hsp25 at Ser 86 and no in vitro binding to 14-3-3 was detectable, stress-dependent disaggregation of endogenous Hsp25 complexes is impared and kinetics of arsenite-dependent, H 2 O 2 -dependent, and sublethal heat shock-induced insolubilization of Hsp25 is delayed. Similarly, green fluorescent protein-tagged Hsp25 shows retarded subcellular accumulation into stress granules in MK2-deficient cells after arsenite treatment. Decreased insolubilization of Hsp25 in MK2-deficient cells correlates with increased resistance against arsenite, H 2 O 2 , and sublethal heat shock treatment and with decreased apoptosis. In contrast, after severe, lethal heat shock MK2-deficient embryonic fibroblasts cells show fast and complete insolubilization of Hsp25 independent of MK2 and no increased stress resistance. Hence, MK2-dependent formation of insoluble stress granules and irreversible cell damage by oxidative stresses and sublethal heat shock correlate and only upon severe, lethal heat shock MK2-independent processes could determine insolubilization of Hsp25 and are more relevant for cellular stress damage.
Septin 7 (SEPT7) has been described to be essential for successful completion of cytokinesis in mouse fibroblasts, and Sept7-deficiency in fibroblasts constitutively results in multinucleated cells which stop proliferation. Using Sept7flox/floxfibroblasts we generated a cellular system, where the cytokinetic defects of Cre-mediated deletion of the Sept7 gene can be rescued by ectopically expressed doxycycline-inducible wild type SEPT7. Using this system, we analyzed the ability of SEPT7-mutants with alterations in their GTPase domain-dependent dimerization to prevent multinucleation and rescue proliferation. Although biochemical analysis of the mutants demonstrates differences in homo- and/or hetero-polymerization, in GTP-binding and/or GTPase activities, all analyzed mutants were able to rescue the cytokinesis phenotype of Sept7flox/floxfibroblasts associated with Cre-mediated deletion of endogenous Sept7. These findings indicate that the ability of septins to assemble into well-defined SEPT7-dimerization dependent native filaments is dispensable for cytokinesis in fibroblasts and opens the way to search for other mechanisms of the involvement of SEPT7 in cytokinesis.
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