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Cell-cycle-regulated phosphorylation of cAMP response element-binding protein: identification of novel phosphorylation sites.

ABSTRACT: We report that the cAMP response element binding protein (CREB) undergoes cell-cycle-regulated phosphorylation. In human amnion FL cells, CREB was expressed as two forms with different molecular masses, 45 and 45.5 kDa. Although asynchronous cells contained predominantly the 45 kDa forms, this form shifted to 45.5 kDa when the cells were synchronized with the early S-phase. Furthermore the expression of the 45.5 kDa band was increased when cells were treated with okadaic acid, confirming that the 45.5 kDa band was a phosphorylated form of the 45 kDa band. Mutation analysis indicated that neither Ser133, the target of cAMP-dependent protein kinase and calcium calmodulin kinase, nor Ser129, the target of glycogen synthetase kinase 3, was responsible for the expression of the 45.5 kDa band, but that Ser108, Ser111 and Ser114, located in a region matching the consensus sequence for the casein kinase II target, were required. A mutant in which Ser111 and Ser114 were each replaced by a glutamic residue, mimicking a phosphorylated state, had a higher activation potential in cAMP response element-mediated transcription. These results strongly suggest that the casein kinase II target region is involved in cell cycle-regulated phosphorylation of the CREB protein and also in transcriptional enhancement.

SUBMITTER: Saeki K

PROVIDER: S-EPMC1220023 | biostudies-other | 1999 Feb

REPOSITORIES: biostudies-other

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Project description:GH and GH receptors are expressed throughout life, and GH elicits a diverse range of responses, including growth and altered metabolism. It is therefore important to understand the full spectrum of GH signaling pathways and cellular responses. We applied mass spectrometry-based phosphoproteomics combined with stable isotope labeling with amino acids in cell culture to identify proteins rapidly phosphorylated in response to GH in 3T3-F442A preadipocytes. We identified 132 phosphosites in 95 proteins that exhibited rapid (5 or 15 min) GH-dependent statistically significant increases in phosphorylation by more than or equal to 50% and 96 phosphosites in 46 proteins that were down-regulated by GH by more than or equal to 30%. Several of the GH-stimulated phosphorylation sites were known (e.g. regulatory Thr/Tyr in Erks 1 and 2, Tyr in signal transducers and activators of transcription (Stat) 5a and 5b, Ser939 in tuberous sclerosis protein (TSC) 2 or tuberin). The remaining 126 GH-stimulated sites were not previously associated with GH. Kyoto Encyclopedia of Genes and Genomes pathway analysis of GH-stimulated sites indicated enrichment in proteins associated with the insulin and mammalian target of rapamycin (mTOR) pathways, regulation of the actin cytoskeleton, and focal adhesions. Akt/protein kinase A consensus sites (RXRXXS/T) were the most commonly phosphorylated consensus sites. Immunoblotting confirmed GH-stimulated phosphorylation of all seven novel GH-dependent sites tested [regulatory sites in proline-rich Akt substrate, 40 kDA (PRAS40), regulatory associated protein of mTOR, ATP-citrate lyase, Na(+)/H(+) exchanger-1, N-myc downstream regulated gene 1, and Shc]). The immunoblot results suggest that many, if not most, of the GH-stimulated phosphosites identified in this large-scale quantitative phosphoproteomics analysis, including sites in multiple proteins in the Akt/ mTOR complex 1 pathway, are phosphorylated in response to GH. Their identification significantly broadens our thinking of GH-regulated cell functions.

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Cell-cycle-regulated phosphorylation of cAMP response element-binding protein: identification of novel phosphorylation sites.

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