Hepatocyte growth factor/scatter factor is a multifunctional factor that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Mettyrosine kinase. Upon stimulation with hepatocyte growth factor, the Met ␤ subunit becomes highly phosphorylated on tyrosine residues, one of which, tyrosine 1356 within the carboxyl terminus, is crucial for dissociation, motility, and branching tubule formation in Madin-Darby canine kidney epithelial cells. Tyrosine 1356 forms a multisubstrate binding site for the Grb2 and Shc adaptor proteins, the p85 subunit of phosphatidylinositol 3-kinase, phospholipase C␥, and a phosphatase, SHP2. To investigate additional signaling molecules that are activated by the Met receptor, we have identified hepatocyte growth factor-induced phosphoproteins in tubular epithelial cells. We have established that proteins of 100 -130 kDa are highly phosphorylated following stimulation of epithelial cells and that one of these is the Grb2-associated binding protein Gab1, a possible insulin receptor substrate-1-like signal transducer. We show that Gab1 is the major substrate for the Met kinase in vitro and in vivo. Association of Gab1 with Met requires a functional Grb2 binding site involving tyrosine 1356 and to a lesser extent tyrosine 1349. Met receptor mutants that fail to induce branching tubulogenesis are impaired in their ability to interact with Gab1, suggesting that Gab1 may play a role in these processes.

The Tpr-Met oncoprotein, which is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement, consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. We have previously demonstrated that a single tyrosine residue in the carboxyl terminus, Tyr 489 , is highly phosphorylated and is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and for the association of Tpr-Met with the Grb2 adaptor protein and phosphatidylinositol 3-kinase. We show here that Tyr 489 is also required for association of Tpr-Met with phospholipase C␥ and the tyrosine phosphatase, SHPTP2/Syp. To distinguish which of these substrates are required for cell transfor-

IntroductionCD33 is a 67 kDa type 1 transmembrane sialoglycoprotein and founding member of a rapidly evolving immunoglobulin superfamily subset of sialic acid-binding immunoglobulin-related lectins (siglecs; siglec-3). 1,2 CD33 is characterized by the presence of 2 conserved immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motifs in the cytoplasmic region, a property that is shared with all CD33-related siglecs discovered so far. 2 If phosphorylated upon treatment with pervanadate or anti-CD33 antibody plus crosslinking secondary antibody, these tyrosine motifs recruit and activate Src homology-2 (SH2) domain-containing tyrosine phosphatases (SHP-1 and SHP-2). [3][4][5] Physiologically, CD33 expression is restricted to early multilineage hematopoietic progenitors, myelomonocytic precursors, and more mature myeloid cells, but it is absent on normal pluripotent hematopoietic stem cells. [6][7][8] However, 85% to 90% of adult and pediatric cases of acute myeloid leukemia (AML) express CD33. 8,9 Therefore, CD33 has gained clinical importance as a suitable tumor-associated antigen and target for antibody-based AML therapies. 10 Promising clinical results have been obtained with gemtuzumab ozogamicin (GO; CMA-676; Mylotarg; Wyeth Pharmaceuticals, Philadelphia, PA), an immunoconjugate consisting of a humanized immunoglobulin G 4 (IgG 4 ) anti-CD33 monoclonal antibody (hP67.6) joined to a derivative of the cytotoxic drug calicheamicin-␥ 1 , N-acetyl-␥-calicheamicin dimethyl hydrazide. 11 Although some unconjugated anti-CD33 antibodies may have limited cytoreductive activity when used as monotherapy in patients, 12,13 hP67.6 is believed to function primarily as a carrier to facilitate cellular uptake of the calicheamicin-␥ 1 derivative, which is then cleaved intracellularly in an acid compartment, presumably inside lysosomes, and subsequently induces DNA damage and eventually cell death. 14 This putative mechanism of action of GO still needs firm experimental confirmation but implies a pivotal role of both the number of CD33 molecules expressed on the cell surface and the rate of internalization of CD33 following GO binding for the cytotoxic effect. However, little is known about the importance of the quantity of CD33 expression for GO-induced cytotoxicity. Although multiple studies have confirmed that binding of bivalent and multivalent anti-CD33 antibodies results in CD33 internalization in both CD33 ϩ hematopoietic cell lines and primary AML blast cells, 15-18 some authors have argued that cytotoxic effects of GO are achieved in the absence of CD33 expression. 19 In addition, neither the endocytic process by which CD33 delivers antibody to the cytosol nor the necessity of CD33 endocytosis for GO-induced cytotoxicity has been established.The primary objective of the present study was therefore to investigate the quantitative relationship between CD33 expression and the requirement for CD33 internalization in GO-mediated cytotoxicity. To accomplish this, lentivirus-mediated gene transfer was used to manipulate...