Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

Masaaki Masuhara, Kenji Nagao, Mitsuo Nishikawa, Mika Sasaki, Akihiko Yoshimura, Masatake Osawa

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1+c-kit+Lin-) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1+c-kit+Lin- stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)697-703
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume268
Issue number3
DOIs
Publication statusPublished - 2000 Feb 24
Externally publishedYes

Fingerprint

Space time adaptive processing
src Homology Domains
Cloning
Molecular Cloning
Stem cells
Stem Cells
Tyrosine
Hematopoietic Stem Cells
Bone Marrow Cells
Proteins
Proto-Oncogene Proteins c-kit
Leukemia Inhibitory Factor
Two-Hybrid System Techniques
Cells
Myeloid Cells
Gene Library
Cell Differentiation
Assays
Bone
Complementary DNA

Keywords

  • PH domain
  • Protein tyrosine kinase
  • SH2 domain
  • Signal transduction
  • Stem cell

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains. / Masuhara, Masaaki; Nagao, Kenji; Nishikawa, Mitsuo; Sasaki, Mika; Yoshimura, Akihiko; Osawa, Masatake.

In: Biochemical and Biophysical Research Communications, Vol. 268, No. 3, 24.02.2000, p. 697-703.

Research output: Contribution to journalArticle

Masuhara, Masaaki ; Nagao, Kenji ; Nishikawa, Mitsuo ; Sasaki, Mika ; Yoshimura, Akihiko ; Osawa, Masatake. / Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains. In: Biochemical and Biophysical Research Communications. 2000 ; Vol. 268, No. 3. pp. 697-703.
@article{41a060f54c8f4bcc9e8ebfb8b9519542,
title = "Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains",
abstract = "To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1+c-kit+Lin-) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1+c-kit+Lin- stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells. (C) 2000 Academic Press.",
keywords = "PH domain, Protein tyrosine kinase, SH2 domain, Signal transduction, Stem cell",
author = "Masaaki Masuhara and Kenji Nagao and Mitsuo Nishikawa and Mika Sasaki and Akihiko Yoshimura and Masatake Osawa",
year = "2000",
month = "2",
day = "24",
doi = "10.1006/bbrc.2000.2223",
language = "English",
volume = "268",
pages = "697--703",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

AU - Masuhara, Masaaki

AU - Nagao, Kenji

AU - Nishikawa, Mitsuo

AU - Sasaki, Mika

AU - Yoshimura, Akihiko

AU - Osawa, Masatake

PY - 2000/2/24

Y1 - 2000/2/24

N2 - To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1+c-kit+Lin-) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1+c-kit+Lin- stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells. (C) 2000 Academic Press.

AB - To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1+c-kit+Lin-) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1+c-kit+Lin- stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells. (C) 2000 Academic Press.

KW - PH domain

KW - Protein tyrosine kinase

KW - SH2 domain

KW - Signal transduction

KW - Stem cell

UR - http://www.scopus.com/inward/record.url?scp=0034708227&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034708227&partnerID=8YFLogxK

U2 - 10.1006/bbrc.2000.2223

DO - 10.1006/bbrc.2000.2223

M3 - Article

VL - 268

SP - 697

EP - 703

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 3

ER -