pervanadate and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

STAT5b (signal transducer and activator of transcription 5b) is a key mediator of the effects of plasma GH pulses on male-specific liver gene expression. STAT5b is activated in liver cells in vivo by physiological pulses of GH and then is rapidly deactivated. Investigation of the cellular events involved in this activation/deactivation cycle using the rat liver cell line CWSV-1 established that a brief exposure to GH and the associated activation of JAK2 (Janus kinase 2) tyrosine kinase activity are both necessary and sufficient to initiate all of the downstream steps associated with STAT5b activation by tyrosine phosphorylation and the subsequent deactivation of both JAK2 kinase and STAT5b. JAK2 signaling to STAT5b at the conclusion of a GH pulse could be sustained by the protein synthesis inhibitor cycloheximide or by the proteasome inhibitor MG132, indicating that termination of this JAK2-catalyzed STAT activation loop requires synthesis of a labile or GH-inducible protein factor and is facilitated by the proteasome pathway. This factor may be a phosphotyrosine phosphatase, since the phosphatase inhibitor pervanadate both sustained GH pulse-induced JAK2 signaling to STAT5b and blocked the rapid deactivation of phosphorylated STAT5b (t(1/2) = 8.8 +/- 0.9 min) seen in its absence. Finally, the serine kinase inhibitor H7 blocked down-regulation of JAK2 signaling to STAT5b in a manner that enabled cells to respond to a subsequent GH pulse without the need for the approximately 3-h interpulse interval normally required for full recovery of GH pulse responsiveness. Termination of GH pulse-induced STAT5b signaling is thus a complex process that involves multiple biochemical events. These are proposed to include the down-regulation of JAK2 signaling to STAT5b via a cycloheximide- and H7-sensitive step, proteasome-dependent degradation of a key component or regulatory factor, and dephosphorylation leading to deactivation of the receptor-kinase signaling complex and its STAT5b substrate via the action of a phosphotyrosine phosphatase.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cell Line; Cycloheximide; Cysteine Endopeptidases; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Growth Hormone; Janus Kinase 2; Leupeptins; Liver; Milk Proteins; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Receptors, Somatotropin; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Vanadates

The suppression of male-specific, GH pulse-induced, liver transcription in adult female rats has been linked to the down-regulation of STAT5b activation by the female plasma pattern of near-continuous GH exposure. The mechanism underlying this down-regulation was studied in the rat liver cell line CWSV-1, where continuous GH suppressed the level of activated (tyrosine- phosphorylated) STAT5b to approximately 10-20% of the maximal GH pulse-induced STAT5b signal within 3 h. In contrast to the robust JAK2 kinase-dependent STAT5b activation loop that is established by a GH pulse, JAK2 kinase signaling to individual STAT5b molecules was found to be short lived in cells treated with GH continuously. Moreover, maintenance of the low-level STAT5b signal required ongoing protein synthesis and persisted for at least 7 days provided that GH was present in the culture continuously. Increased STAT5b DNA-binding activity was observed in cells treated with the proteasome inhibitor MG132, suggesting that at least one component of the GH receptor (GHR)-JAK2-STAT5b signaling pathway becomes labile in response to continuous GH treatment. The phosphotyrosine phosphatase inhibitor pervanadate fully reversed the down-regulation of STAT5b DNA-binding activity in continuous GH-treated cells by a mechanism that involves both increased STAT5b activation and decreased STAT5b dephosphorylation. Moreover, the requirement for ongoing GH stimulation and active protein synthesis to maintain STAT5b activity in continuous GH-treated cells were both eliminated by pervanadate treatment, suggesting that phosphotyrosine dephosphorylation may be an obligatory first step in the internalization/degradation pathway for the GHR-JAK2 complex. Finally, the sustaining effect of the serine kinase inhibitor H7 on GH pulse-induced JAK2 signaling to STAT5b was not observed in continuous GH-treated cells. These findings suggest a model where continuous GH exposure of liver cells down-regulates the STAT5b pathway by a mechanism that involves enhanced dephosphorylation of both STAT5b and GHR-JAK2, with the latter step leading to increased internalization/degradation of the re-ceptor-kinase complex.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cells, Cultured; Cycloheximide; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Down-Regulation; Electrophoresis; Female; Genistein; Growth Hormone; Growth Inhibitors; Janus Kinase 2; Leupeptins; Liver; Male; Milk Proteins; Protein Synthesis Inhibitors; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Signal Transduction; STAT5 Transcription Factor; Staurosporine; Trans-Activators; Vanadates