Novel role of WD40 and SOCS box protein-2 in steady-state distribution of granulocyte colony-stimulating factor receptor and G-CSF-controlled proliferation and differentiation signaling.

Signals induced by granulocyte colony-stimulating factor (G-CSF), the major cytokine involved in neutrophil development, are tightly controlled by ligand-induced receptor internalization. Truncated G-CSF receptors (G-CSF-Rs) that fail to internalize show sustained proliferation and defective differentiation signaling. Steady-state forward routing also determines cell surface levels of cytokine receptors, but mechanisms controlling this are poorly understood. Here, we show that WD40 and suppressor of cytokine signaling (SOCS) box protein-2 (Wsb-2), an SOCS box-containing WD40 protein with currently unknown function, binds to the COOH-terminal region of G-CSF-R. Removal of this region did not affect internalization, yet resulted in increased membrane expression of G-CSF-R and enhanced proliferation signaling at the expense of differentiation induction. Conversely, Wsb-2 binding to the G-CSF-R reduced its cell surface expression and inhibited proliferation signaling. These effects depended on the SOCS box involved in ubiquitylation and on cytosolic lysines of G-CSF-R and imply a major role for ubiquitylation through the G-CSF-R C-terminus in forward routing of the receptor. Importantly, the Wsb-2 gene is commonly disrupted by virus integrations in mouse leukemia. We conclude that control of forward routing of G-CSF-R is essential for a balanced response of myeloid progenitors to G-CSF and suggest that disturbance of this balance may contribute to myeloid leukemia.

Multiple pathways contribute to the hyperproliferative responses from truncated granulocyte colony-stimulating factor receptors.

Mutations in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene leading to a truncated protein have been identified in a cohort of neutropenia patients highly predisposed to acute myeloid leukemia. Such mutations act in a dominant manner resulting in hyperproliferation but impaired differentiation in response to G-CSF. This is due, at least in part, to defective internalization and loss of binding sites for several negative regulators, leading to sustained receptor activation. However, those signaling pathways responsible for mediating the hyperproliferative function have remained unclear. In this study, analysis of an additional G-CSF-R mutant confirmed the importance of residues downstream of Box 2 as important contributors to the sustained proliferation. However, maximal proliferation correlated with the ability to robustly activate signal transducer and activator of transcription (STAT) 5 in a sustained manner, whereas co-expression of dominant-negative STAT5, but not dominant-negative STAT3, was able to inhibit G-CSF-stimulated proliferation from a truncated receptor. Furthermore, a Janus kinase (JAK) inhibitor also strongly reduced the proliferative response, whereas inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) or phosphatidylinositol (PI) 3-kinase reduced proliferation to a lesser degree. These data suggest that sustained JAK2/STAT5 activation is a major contributor to the hyperproliferative function of truncated G-CSF receptors, with pathways involving MEK and PI 3-kinase playing a reduced role.

The SH2 domain-containing protein tyrosine phosphatase SHP-1 is induced by granulocyte colony-stimulating factor (G-CSF) and modulates signaling from the G-CSF receptor.

The SH2 domain-containing protein tyrosine phosphatase SHP-1 is expressed widely in the hematopoietic system. SHP-1 has been shown to negatively control signal transduction from many cytokine receptors by direct docking to either the receptor itself, or to members of the Jak family of tyrosine kinases which are themselves part of the receptor complex. Motheaten and viable motheaten mice, which are deficient in SHP-1, have increased myelopoiesis and show an accumulation of morphologically and phenotypically immature granulocytes, suggesting a role for SHP-1 in granulocytic differentiation. Here, we report that SHP-1 protein levels are up-regulated during the granulocyte colony-stimulating factor (G-CSF)-mediated granulocytic differentiation of myeloid 32D cells. Enforced expression of SHP-1 in these cells leads to decreased proliferation and enhanced differentiation, while introduction of a catalytically inactive mutant produces increased proliferation and results in a delay of differentiation. In vitro binding revealed that the SH2 domains of SHP-1 are unable to associate directly with tyrosine-phosphorylated G-CSF receptor (G-CSF-R). Furthermore, over-expression of SHP-1 in Ba/F3 cells expressing a G-CSF-R mutant lacking all cytoplasmic tyrosines also inhibited proliferation. Together, these data suggest that SHP-1 directly modulates G-CSF-mediated responses in hematopoietic cells via a mechanism that does not require docking to the activated G-CSF-R.

Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Absorption of different amino acids by an intestinal preparation from normal rats and from rats exposed to supralethal X-irradiation.

Absorption of different amino acids [lysine, alpha-amino isobutyrate (AIB), methionine, alanine, glycine] was studied in an intestinal in vivo preparation from normal and X-irradiated rats (1 and 3 days after 2000 R). Absorption of amino acids under loading and in presence of other amino acids was also followed. Lysine and AIB absorption follow a saturation kinetics with a maximal velocity of 35 and 85.6 micronmoles/min and a Kt of 10 and 67 micronmoles for lysine and AIB respectively. Interference in absorption exists not only between amino acids of the same class, but also by lysine on neutral amino acids. One day after irradiation, absorption is normal or even enhanced, although active transport appears impaired as indicated by the increase in Kt. Three days after exposure absorption, particularly the active one, is severely depressed. This is indicated by the low Vm and high Kt as well as by the fact that loading has little influence on absorption.