Development of Efficient Two-Step Deprotection Methodology for Dimethyl-Protected Phosphoamino Acid-Containing Peptide Resins and Its Application to the Practical Synthesis of Phosphopeptides

Otaka, Akira; Miyoshi, Kengo; Kaneko, M.; Tamamura, Hirokazu; Fujii, Nobutaka; Nomizu, Motoyoshi; Burke, Terrence R.; Roller, Peter P.

doi:10.1021/jo00118a011

Cited by 28 publications

(14 citation statements)

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“…The purity of the peptides was confirmed by analytical high performance liquid chromatography. The identity of the peptides was confirmed by a Scion API IIIE triple quadruple ion spray mass spectrometer (35).…”

Section: Methodsmentioning

confidence: 99%

Cell Binding Sequences in Mouse Laminin α1 Chain

Nomizu¹,

Kuratomi²,

Malinda³

et al. 1998

Journal of Biological Chemistry

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144

148

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Section: Methodsmentioning

confidence: 99%

Cell Binding Sequences in Mouse Laminin α1 Chain

Nomizu¹,

Kuratomi²,

Malinda³

et al. 1998

Journal of Biological Chemistry

Self Cite

144

148

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“…Purity of the peptides was confirmed by analytical high performance liquid chromatography. All synthetic peptides were characterized by a Sciex API IIIE triple quadrupole ion spray mass spectrometer (27). Mouse laminin-1 was prepared from the Engelbreth-Holm-Swarm tumor as described previously (28).…”

Section: Methodsmentioning

confidence: 99%

Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening

Nomizu¹,

Kuratomi²,

Song³

et al. 1997

Journal of Biological Chemistry

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107

101

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Laminin-1, a major component of basement membranes, consists of three different chains designated ␣1, ␤1, and ␥1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin ␥1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the ␥1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-␣ 2 ␤ 1 integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin ␥2 chain. These results suggest that the specific sequences on the laminin ␥1 chain are biologically active and interact with distinct cell surface receptors.Laminin-1, a major component of the basement membrane matrix, has multiple biological activities including promotion of cell adhesion, spreading, growth, neurite outgrowth, tumor metastasis, and collagenase IV secretion (1-4). There are at least 11 isoforms of laminin, each consisting of three different chains (5). The most extensively characterized laminin, laminin-1 (M r ϭ 900,000) from the mouse Engelbreth-Holm-Swarm tumor consists of ␣1, ␤1, and ␥1 chains, which assemble into a triple-stranded cross-like structure with a coiled-coil domain at the long arm (6).Several active sequences on laminin-1 have been identified using proteolytic fragments, recombinant proteins and synthetic peptides (7,8). The YIGSR sequence located on the ␤1 chain promotes cell adhesion and migration, and inhibits angiogenesis and tumor metastasis (9 -11). The PDSGR and F-9 (RYVVLPR) sequences located on the ␤1 chain also promote cell adhesion (12, 13). An IKVAV sequence located on the C-terminal end of the long arm of the ␣1 chain promotes cell adhesion, neurite outgrowth, experimental metastasis, collagenase IV activity, angiogenesis, plasminogen activator activation, cell growth, and tumor growth (14 -18). Recently, we identified five cell binding sequences in the mouse laminin ␣1 chain C-terminal globular domain, G domain (positions 2111-3060), by systematic peptide screening using overlapping peptide-polystyrene beads and free peptides.Although the laminin ␥1 chain is present in most laminin isoform...

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“…Norleucines (Z) were used instead of methionines (M) as described previously (19). Boc-based techniques were utilized for the preparation of the phosphopeptide possessing substitution of phosphotyrosine (pY) or difluoroPmp for tyrosine in the two YZPZ motifs (20,21). The peptides were purified by preparative reverse phase HPLC.…”

Section: Methodsmentioning

confidence: 99%

“…tion of untreated 3T3L1 adipocytes, and the mixture was immunoprecipitated with anti-p85 antibody (20,30). The immunoabsorbed PI3K was further phosphorylated by adding ATP and manganese and then subjected to in vitro association experiments with GST-14-3-3␤ protein.…”

Section: Fig 2 In Vitro Association Of 14-3-3␤ Protein With Irs-1 mentioning

confidence: 99%

14-3-3β Protein Associates with Insulin Receptor Substrate 1 and Decreases Insulin-stimulated Phosphatidylinositol 3′-Kinase Activity in 3T3L1 Adipocytes

Kosaki

Yamada

Suga

et al. 1998

Journal of Biological Chemistry

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The 14-3-3 protein family has been implicated in growth factor signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3␤ antibody at the basal condition. 100 nM insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nM wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3␤ fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3-kinase (PI3K) is supposed to complex either 14-3-3␤-IRS-1 or IRS-1. The 14-3-3␤-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3␤ and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3␤ protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.Insulin promotes the rapid autophosphorylation of its receptor ␤-subunits and tyrosine phosphorylation of several cytoplasmic proteins such as Shc, pp60, Gab-1, and insulin receptor substrate (IRS) 1 1 and 2. IRS-1 plays a central role in insulin signaling. The protein contains a pleckstrin homology domain, a phosphotyrosine-binding domain that binds to NPXY motif of the insulin receptor ␤-subunit and multiple tyrosine residues that are potential phosphorylation sites. The tyrosine-phosphorylated IRS-1 associates with several Src homology 2 domaincontaining proteins including Grb-2, SHP-2, Nck, Fyn, and the 85-kDa subunit (p85) of phosphatidylinositol 3Ј-kinase (PI3K).Associations of these molecules with IRS-1 are believed to further activate downstream signaling systems, including mitogen-activated protein kinase and PI3K cascades, that promote mitogenic and metabolic effects of insulin (for review see Refs. 1 and 2).14-3-3 protein family, first discovered as acidic proteins in the brain, is highly conserved in animals and plants. At least nine mammalian isoforms have been identified, and six are expressed ubiquitously (3). Originally, 14-3-3 proteins have been shown to be functional as regulators of tryptophan and tyrosine hydroxylases as well as protein kinase C (for review see Ref. 4). However, recently 14-3-3 proteins were found to associate oncogene products, including Raf-1 (5-11), Bcr-Abl, Bcr (12, 13), polyoma middle T antigen (14), and cell cycle control proteins such as Cdc25 phosphatases (15). The interaction of Raf with 14-3-3 leads to Raf activation in several in vivo systems (5,7,8). Moreover, the importance of 14-3-3 proteins in ...

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Development of Efficient Two-Step Deprotection Methodology for Dimethyl-Protected Phosphoamino Acid-Containing Peptide Resins and Its Application to the Practical Synthesis of Phosphopeptides

Cited by 28 publications

References 1 publication

Cell Binding Sequences in Mouse Laminin α1 Chain

Cell Binding Sequences in Mouse Laminin α1 Chain

Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening

14-3-3β Protein Associates with Insulin Receptor Substrate 1 and Decreases Insulin-stimulated Phosphatidylinositol 3′-Kinase Activity in 3T3L1 Adipocytes

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