Signal transduction via the CD38/NAD+ glycohydrolase.
The human cell surface CD38 molecule is a 46-kDa type-II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long Cys-rich C-terminal extracellular one. We previously demonstrated that an ecto-form NAD+ glycohydrolase (NADase) activity induced by all-trans retinoic acid in HL-60 cells is due to the extracellular domain of CD38. In the present study, we investigated a possible signal transduction mediated through CD38 in the retinoic acid-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (mAbs). The addition of selected anti-CD38 mAbs to the cells induced rapid tyrosine phosphorylation of the cellular proteins with the molecular weights of 120,000, 87,000 and 77,000; the phosphorylated 120-kDa protein was identified as the c-cbl proto-oncogene product, p120c-cbl. Furthermore, the phosphorylated p 120c-cbl associated with the 85-kDa subunit of phosphatidylinositol 3-kinase. To determine the relationship between the amino acid sequence responsible for the NADase activity and epitopes recognized by the stimulatory mAbs, we produced its carboxy-terminal deletion mutants in COS-7 cells. The mutants with less than 15 amino acids deleted from the carboxyl terminus of the 300-amino acid wild-type molecule still maintained NADase activity, but those with more than 27 amino acids deleted did not. Introduction of site-directed mutation of a cysteine residue (Cys275), located in the 273-285 sequence, completely abolished the NADase activity. These CD38 mutants were also used for an epitope mapping of anti-CD38 mAbs. All the epitopes recognized by the mAbs inducing the tyrosine phosphorylation were mapped on the same Cys275-containing sequence of 273-285. Thus, the discrete carboxy-terminal sequence not only plays a key role in its ecto-NADase activity, but also contains the epitopes of the agonistic anti-CD38 mAbs for the transmembrane signaling. We also found that the agonistic mAbs markedly potentiate superoxide generation induced by the stimulation of G protein-coupled chemotactic receptors. Our results suggested that the stimulation of CD38 might generate an accessory signal(s) to enhance the G protein-mediated signaling, probably though the protein-tyrosine phosphorylation.
Kontani
K
K
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Kukimoto
I
I
Kanda
Y
Y
Inoue
S
S
Kishimoto
H
H
Hoshino
S
S
Nishina
H
H
Takahashi
K
K
Hazeki
O
O
Katada
T
T
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D001048:Aplysia / Q000201:enzymology; D001665:Binding Sites; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D003545:Cysteine; D018604:Epitope Mapping; D018922:HL-60 Cells; D006801:Humans; D008562:Membrane Glycoproteins; D009000:Monocytes / Q000166:cytology; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D018000:Receptors, Peptide / Q000378:metabolism; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; C000715470:MAS1 protein, human; D008562:Membrane Glycoproteins; D011506:Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D013481:Superoxides; D014443:Tyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1; C067419:CBL protein, human; D003545:Cysteine