Single amino acid substitution, from Glu1025 to Asp, of the fps oncogenic protein causes temperature sensitivity in transformation and kinase activity

Chen, LiHow; Hatada, Eunice N.; Wheatley, William; Wen-Hwa, Lee

doi:10.1016/0042-6822(86)90172-8

Cited by 8 publications

(3 citation statements)

References 34 publications

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“…The temperature sensitivity of a normal laboratory variant of FSV is associated with substitution of a few residues downstream from the AX14 locus, Glu-1025 to Asp (Fig. 7), although a second mutation in the amino-terminal fps domain also contributes to temperature sensitivity (6). Temperature-sensitive v-abl mutations isolated in an E. coli expression system were found exclusively in the corresponding region of this gene (21).…”

Section: Discussionmentioning

confidence: 99%

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus

Johnson

Stone

1990

J Virol

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We analyzed linker insertion mutations throughout the 3' region of the v-fps gene of Fujinami sarcoma virus to identify tyrosine kinase transforming protein (P13VflK-fPs) determinants that are important for catalysis and transforming activity and, in particular, to define residues that participate in substrate selection. Mutations that encode kinase-active, transformation-defective v-fps alleles were recovered, defining sites in the transforming protein that may normally facilitate kinase-substrate interaction. Additionally, one region within the catalytic domain of the transforming protein (amino acid residues 1012 to 1020) that tolerates peptide insertions without loss of transforming activity was discovered, although the insertion mutations in this region of v-fps exhibited qualitatively abnormal transforming function. Transformed rat cell lines that express these mutations displayed unusual phenotypes, including giant cells and cells with an extremely fusiform shape. Furthermore, the insertion mutations in this region were temperature sensitive, transformed cells assumed a flat morphology, cellular protein phosphotyrosine was reduced, and the kinase activity of the transforming protein was decreased when cells were incubated at 40.5°C. Point mutations that specify the ancestral chicken c-fps sequence in the insertion-tolerant region were also introduced into v-fps. These back mutations led to a modest decrease in kinase activity, decreased tumorigenic potential in chickens, and an unexpected increase in transforming activity in rat cells. These results indicate that the insertion-tolerant region of P130gag-fPs influences the biologic activity and thermostability of the kinase.

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Section: Discussionmentioning

confidence: 99%

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus

Johnson

Stone

1990

J Virol

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“…It is likely that the Ala --Gly substitution reduces the stability of the reaction complex and thus accounts for the temperature sensitivity. The mutation sites of other known ts mutants of PTK oncogenes vary broadly, mapping mostly to the large lobe (31)(32)(33)(34). Each of the three v-src ts mutants NY68, NY72-4, and PA104 contains two mutations including a common Valh461 -Met mutation (35,36), at a position corresponding to Pro236 of cAPK, which is involved in its high-affinity substrate binding site interaction (9).…”

Section: Resultsmentioning

confidence: 99%

Ala-->Gly mutation in the putative catalytic loop confers temperature sensitivity on Ros, insulin receptor, and insulin-like growth factor I receptor protein-tyrosine kinases.

Chen

Hanafusa

Wang

1994

Proc. Natl. Acad. Sci. U.S.A.

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Temperature-sensitive mutations in the avian sarcoma virus UR2 oncogene ros, encoding a receptor proteintyrosine kinase (PTK), were identified. The Ala3 -Gly change mapping within the highly conserved RDLAARN motif in the Ros kinase domain was responsible for the temperaturesensitive phenotype. Based on the sequence homology of all known protein kinases and the crystalline structure of the cAMP-dependent protein kinase, this conserved region probably represents the PTK catalytic loop. The same mutation when introduced into the human insulin and insulin-like growth factor I receptors made these PTKs temperature sensitive in both biological function and kinase activity. Our results support the presumed catalytic role of this highly conserved sequence in PTKs. Due to its highly conserved nature, we predict that the same mutation would probably confer temperature sensitivity on other PTKs.Protein phosphorylation mediated by tyrosine and serine/ threonine kinases is an important step in many signal transduction pathways, including normal cell growth, embryonic development and morphogenesis, activation of T cells, B cells, and neutrophils, neurotransmitter signaling, metabolic processes, and oncogenesis (1-7). In spite of their enormous diversity of physiological functions, the >100 known protein kinases share a conserved catalytic domain that is characterized by many highly conserved amino acid residues located in 11 major conserved subdomains (8). Most of these residues directly take part in the phosphorylation processes including ATP binding, substrate-peptide interaction, and transfer of the phosphate group (8,9). Recently, analysis of the crystal structure of the catalytic subunit of the cAMPdependent protein kinase (cAPK) revealed two highly conserved loop structures (9). The glycine loop (residues 50-55, GXGXXG) located in the small lobe is responsible for anchoring the ATP moiety, whereas the catalytic loop (residues 165-171, RDLKPEN) in the large lobe is essential for peptide binding and catalysis (9). The corresponding sequences of the two loops in protein-tyrosine kinases (PTKs) are GXGXXG and RDLAARN (for most PTKs) or RDL-RAAN (for src family PTKs) (8, 9). Although the crystal structure of none of the PTK family is known, judging from overall hierarchy of the conserved sequence domains among all protein kinases, it is highly likely that these sequences also play the same role in PTKs. The function of these conserved sequences can be studied by genetic approaches using engineered or spontaneous mutants such as temperaturesensitive (ts) mutants defective in kinase activity. Although it was shown that an Asp -* Asn mutation in the putative catalytic loop of c-Kit and c-Fms resulted in severely diminished kinase activity (10, 11), to our knowledge, no mutations have been mapped to this region that resulted in a ts phenoThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solel...

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“…Comparison of this sequence with the known sequence of the temperature-resistant FSV-2 (19) and the chicken c-fps gene (8) identified two putative ts mutations. Further analysis of recombinants between the ts and temperature-resistant FSV sequences indicates that a change of Glu to Asp in the protein kinase domain, at position 1025, is sufficient for a complete ts phenotype (3).…”

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confidence: 99%

Investigation of the role of P130gag-fps in transformation: generation and use of a temperature-sensitive mutant P130gag-fps

Weinmaster

Hunter

1988

J Virol

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Single amino acid substitution, from Glu1025 to Asp, of the fps oncogenic protein causes temperature sensitivity in transformation and kinase activity

Cited by 8 publications

References 34 publications

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus

Ala-->Gly mutation in the putative catalytic loop confers temperature sensitivity on Ros, insulin receptor, and insulin-like growth factor I receptor protein-tyrosine kinases.

Investigation of the role of P130gag-fps in transformation: generation and use of a temperature-sensitive mutant P130gag-fps

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