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Evolutionary analysis of proline-directed phosphorylation sites in the mammalian growth cone identified using phosphoproteomics.

ABSTRACT: The growth cone is essential for nerve growth and axon regeneration, which directly form and rearrange the neural network. Recently, to clarify the molecular signaling pathways in the growth cone that utilize protein phosphorylation, we performed a phosphoproteomics study of mammalian growth cone membranes derived from the developing rodent brain and identified >?30,000 phosphopeptides from ~?1200 proteins. We found that the phosphorylation sites were highly proline directed and primarily mitogen-activated protein kinase (MAPK) dependent, due to particular activation of c-jun N-terminal protein kinase (JNK), a member of the MAPK family. Because the MAPK/JNK pathway is also involved in axon regeneration of invertebrate model organisms such Caenorhabditis elegans and Drosophila, we performed evolutionary bioinformatics analysis of the mammalian growth cone phosphorylation sites. Although these sites were generally conserved within vertebrates, they were not necessarily conserved in these invertebrate model organisms. In particular, high-frequency phosphorylation sites (>?20 times) were less conserved than low-frequency sites. Taken together, the mammalian growth cones contain a large number of vertebrate-specific phosphorylation sites and stronger dependence upon MAPK/JNK than C. elegans or Drosophila. We conclude that axon growth/regeneration likely involves many vertebrate-specific phosphorylation sites.

SUBMITTER: Igarashi M

PROVIDER: S-EPMC6545026 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Evolutionary analysis of proline-directed phosphorylation sites in the mammalian growth cone identified using phosphoproteomics.

Igarashi Michihiro M Okuda Shujiro S

Molecular brain 20190531 1

The growth cone is essential for nerve growth and axon regeneration, which directly form and rearrange the neural network. Recently, to clarify the molecular signaling pathways in the growth cone that utilize protein phosphorylation, we performed a phosphoproteomics study of mammalian growth cone membranes derived from the developing rodent brain and identified > 30,000 phosphopeptides from ~ 1200 proteins. We found that the phosphorylation sites were highly proline directed and primarily mitoge ...[more]

PMID: 31151465

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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.

Dataset Information

Evolutionary analysis of proline-directed phosphorylation sites in the mammalian growth cone identified using phosphoproteomics.

Publications

Evolutionary analysis of proline-directed phosphorylation sites in the mammalian growth cone identified using phosphoproteomics.

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