The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels

Margaret M. Chou, Jorge E. Fajardo, Hidesaburo Hanafusa

Research output: Contribution to journalArticle

78 Citations (Scopus)

Abstract

We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-γ. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nek with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nek may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.

Original languageEnglish (US)
Pages (from-to)5834-5842
Number of pages9
JournalMolecular and Cellular Biology
Volume12
Issue number12
StatePublished - Dec 1992
Externally publishedYes

Fingerprint

Phosphotyrosine
Fibroblasts
Protein-Serine-Threonine Kinases
Glutathione Transferase
Oncogenes
Protein-Tyrosine Kinases
Tyrosine
ras GTPase-Activating Proteins
Phosphatidylinositol 3-Kinase
Proteins
Vanadates
src Homology Domains
Type C Phospholipases
Substrate Specificity
Agar
Cell Line
Nck protein
Neoplasms

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels. / Chou, Margaret M.; Fajardo, Jorge E.; Hanafusa, Hidesaburo.

In: Molecular and Cellular Biology, Vol. 12, No. 12, 12.1992, p. 5834-5842.

Research output: Contribution to journalArticle

@article{9f68fb6340f9439495e808b7a6a2fa33,
title = "The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels",
abstract = "We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-γ. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nek with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nek may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.",
author = "Chou, {Margaret M.} and Fajardo, {Jorge E.} and Hidesaburo Hanafusa",
year = "1992",
month = "12",
language = "English (US)",
volume = "12",
pages = "5834--5842",
journal = "Molecular and Cellular Biology",
issn = "0270-7306",
publisher = "American Society for Microbiology",
number = "12",

}

TY - JOUR

T1 - The SH2- and SH3-containing Nck protein transforms mammalian fibroblasts in the absence of elevated phosphotyrosine levels

AU - Chou, Margaret M.

AU - Fajardo, Jorge E.

AU - Hanafusa, Hidesaburo

PY - 1992/12

Y1 - 1992/12

N2 - We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-γ. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nek with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nek may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.

AB - We have established the human nck sequence as a new oncogene. Nck encodes one SH2 and three SH3 domains, the Src homology motifs found in nonreceptor tyrosine kinases, Ras GTPase-activating protein, phosphatidylinositol 3-kinase, and phospholipase C-γ. Overexpression of human nck in 3Y1 rat fibroblasts results in transformation as judged by alteration of cell morphology, colony formation in soft agar, and tumor formation in nude BALB/c mice. However, overexpression of nck does not induce detectable elevation of the phosphotyrosine content of specific proteins, as is observed for v-crk, another SH2/SH3-containing oncogene. Despite this fact, we demonstrate that Nck retains the ability to bind tyrosine phosphorylated proteins in vitro, using a fusion protein of Nek with glutathione-S-transferase (GST). Moreover, when incubated with lysates prepared from v-src-transformed 3Y1 cells or the nck-overexpressing cell lines, GST-Nck binds to both p60v-src and serine/threonine kinases, respectively. Although phosphotyrosine levels are not elevated in the nck-expressing fibroblasts, vanadate treatment of these cells results in a phosphotyrosine pattern that is altered from the parental 3Y1 pattern, suggestive of a perturbation of indigenous tyrosine kinase pathways. These results suggest the possibility that human nck induces transformation in 3Y1 fibroblasts by virtue of its altered affinity or specificity for the normal substrates of its rat homolog and that Nek may play a role in linking tyrosine and serine/threonine kinase pathways within the cell.

UR - http://www.scopus.com/inward/record.url?scp=0026453352&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026453352&partnerID=8YFLogxK

M3 - Article

VL - 12

SP - 5834

EP - 5842

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 12

ER -