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Regulation of the stress-activated protein kinase pathways in hematopoietic cells Foltz, Ian Nevin


The regulation of the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways in hematopoietic cells is largely uncharacterized. We demonstrated the tyrosine phosphorylation and activation of p38 MAPK by interleukin (IL) -3, granulocytemacrophage colony-stimulating factor (GM-CSF) and Steel locus factor (SLF). We also showed the activation of p38 MAPK is required for the activation of MAPKAP kinase-2 by these cytokines. The activation of JNK often parallels the activation of p38 MAPK. We found that these cytokines also activated the 46 and 55 kDa splice variants of JNK1 and JNK2, and induced threonine phosphorylation of MAPK kinase 4 (MKK4), an upstream activator of JNK. Together, these results demonstrated that p38 MAPK and JNK are activated not only by environmental stresses, but also by hematopoietic growth factors. In contrast, a related cytokine IL-4 failed to activate p38 MAPK, MAPKAP kinase-2, JNK1, JNK2 or MKK4. Therefore, IL-4 was unique in failing to activate any MAPK pathways in hematopoietic cells. Both SLF and GM-CSF activated JNK comparably with similar kinetics, however SLF induced greater phosphorylation of MKK4 than GM-CSF, suggesting the existence of other JNK kinases. A cDNA with homology to MKK4 was identified in the Expressed Sequence Tag database, and the full-length cDNA was cloned using Rapid-Amplification of Cohesive Ends (RACE) PCR. This cDNA encoded a 419 amino acid protein, hereafter termed MKK7, that contained a putative kinase domain. We identified several splice variants of MKK7, and transcripts encoding MKK7 were expressed ubiquitously. Co-expression of MKK7 with either JNK1 or p38a2 MAPK led to the activation of JNK1, but not p38a2 MAPK. Epitope-tagged MKK7 was activated by IL-3, TNFa, UV light, hyperosmolarity, heat shock and anisomycin, but not by IL-4 or EGF. We also showed that endogenous MKK4 and MKK7 were activated by IL-3, TNFa or the Fc receptor for IgG. MKK7 was also activated by constitutively-active Rac and Cdc42 when expressed in HeLa or Ba/F3 cells. In contrast, constitutively-active Ras activated MKK7 in HeLa cells, but not in Ba/F3 cells, demonstrating that signalling from Ras to MKK7 varies between cell types. In conclusion, MKK7 is regulated by diverse stresses and physiological stimuli in hematopoietic cells.

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