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

26 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

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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