The activation of STAT3 by the cytokine receptor gp130 is required for both the G1 to S cell cycle transition and antiapoptosis. We found that Pim-1 and Pim-2 are targets for the gp130-mediated STAT3 signal. Expression of a kinase-defective Pim-1 mutant attenuated gp130-mediated cell proliferation. Constitutive expression of Pim-1 together with c-myc, another STAT3 target, fully compensated for loss of the STAT3-mediated cell cycle progression, antiapoptosis, and bcl-2 expression. We also identified valosine-containing protein (VCP) as a target gene for the Pim-1-mediated signal. Expression of a mutant VCP led cells to undergo apoptosis. These results indicate that Pim-family proteins play crucial roles in gp130-mediated cell proliferation and explain the synergy between Pim and c-Myc proteins in cell proliferation and lymphomagenesis.
Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/α-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.
The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.
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