Transcriptional co-activator p300 maintains basal hepatic gluconeogenesis.

A major cause of fasting hyperglycemia in diabetes mellitus is unregulated hepatic glucose production (HGP). Insulin suppresses HGP by phosphorylating CBP and disassembling the CREB-CBP complex from gluconeogenic genes. p300 is closely related to CBP; but in contrast to CBP, p300 binds constitutively to CREB due to the absence of phosphorylation site found in CBP. In a phosphorylation-competent p300(G442S) knock-in mouse model, we demonstrate that HGP is now exquisitely sensitive to insulin suppression. p300(G422S) and hepatic-deleted p300 mice exhibited significant lower blood glucose levels in the fasted and post-prandial states, indicating a role for p300 in maintaining basal HGP.

Insulin regulation of hepatic gluconeogenesis through phosphorylation of CREB-binding protein.

Hepatic gluconeogenesis is essential for maintenance of normal blood glucose concentrations and is regulated by opposing stimulatory (cyclic adenosine monophosphate, cAMP) and inhibitory (insulin) signaling pathways. The cAMP signaling pathway leads to phosphorylation of cAMP response element-binding (CREB) protein, resulting in recruitment of the coactivators CREB-binding protein (CBP) and p300 and subsequent activation of gluconeogenesis. Insulin signaling leads to phosphorylation of CBP at serine 436, a residue near its CREB-interacting domain, but it is unknown whether this event modulates cAMP signaling. Here, we show in vitro and in 'knock-in' mice that a mutant CBP (S436A) is aberrantly recruited to CREB protein, resulting in inappropriate activation of gluconeogenesis in the fed state and glucose intolerance resulting from increased hepatic glucose production. We propose that insulin signaling may directly regulate many cAMP signaling pathways at the transcriptional level by controlling CBP recruitment.

The role of CBP/p300 interactions and Pit-1 dimerization in the pathophysiological mechanism of combined pituitary hormone deficiency.

CONTEXT: Combined pituitary hormone deficiency (CPHD) in humans is caused by mutations of pituitary-specific transcription factors such as Pit-1. Although many patients with CPHD have an autosomal recessive disorder caused by a Pit-1 DNA-binding mutation, there are a number of reports of mutant Pit-1 molecules that either by prediction or through experimentation bind normally to DNA. OBJECTIVE: The objective of this study was to understand the pathophysiological mechanisms of mutant Pit-1 molecules with intact DNA binding. DESIGN: DNA-binding and functional studies were used to assess five Pit-1 mutations: F135C, R143Q, A158P, K216E, and R271W. RESULTS: In gel-shift studies using well-characterized DNA-binding elements from the GH and prolactin genes, the K126E mutant displayed markedly enhanced Pit-1 dimer binding to either element, whereas the R271W mutant bound with high avidity, but only as a monomer. In contrast, the R143Q mutant was unable to bind these elements, and the F135C and A158P mutants displayed near-normal DNA-binding characteristics. We observed that CBP/p300 bound poorly to the A158P and K216E mutant Pit-1 molecules, but bound normally to the F135C, R143Q, and R271W mutants. In functional assays, CBP/p300 cotransfection with mutant Pit-1 expression vectors resulted in less transactivation of either the GH or prolactin reporter genes. CONCLUSIONS: From these studies, we suggest that CBP/p300 recruitment and Pit-1 dimerization are necessary for Pit-1 target gene activation and are important in the pathogenesis of CPHD.

A novel technique for temporally regulated cell type-specific Cre expression and recombination in the pituitary gonadotroph.

Inducing tissue-specific genetic alterations under temporal control allows for the analysis of gene function in particular cell types at specified points in time. We have generated a system for tetracycline-controlled expression of Cre recombinase in mice using the unique CreTeR vector. The gonadotroph-specific bovine alpha-subunit (Balpha) promoter fragment was subcloned into the CreTeR vector, creating a technique for highly regulated expression of Cre recombinase exclusively in pituitary gonadotrophs. Control of Cre recombinase in the CreTeR vector was demonstrated in LbetaT2 pituitary cell lines, where Cre protein was detected in cells treated with doxycycline, but not in untreated cells. In transgenic mice, Cre was expressed in pituitary gonadotrophs of mice treated with doxycycline, but not in non-pituitary tissues or in transgenic mice not treated with doxycycline. We demonstrated Cre expression in the gonadotroph by immunostaining showing co-localization of Cre recombinase with the beta-subunit of LH (LH-beta). Furthermore, by crossing Balpha/CreTeR with R26R mice, we were able to demonstrate functional recombination within pituitary gonadotrophs, detected by lacZ expression. The Balpha/CreTeR mice described here can be used to study the function of virtually any gene in the gonadotroph; in particular, this will be useful in studying genes, which may have distinct roles in development and in the adult.

Raf kinase inhibitory protein regulates aurora B kinase and the spindle checkpoint.

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.