Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains.
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.
Masuhara
M
M
Institute of Life Science, Kurume University, Aikawamachi 2432-3, Kurume, 839-0861, Japan.
Nagao
K
K
Nishikawa
M
M
Sasaki
M
M
Yoshimura
A
A
Osawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D000950:Antigens, Ly / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D018952:Antigens, CD34; D000950:Antigens, Ly; D017931:DNA Primers; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D012333:RNA, Messenger; D019009:Proto-Oncogene Proteins c-kit