Proc. Natl. Acad. Sci. USA Vol. 95, pp. 7927–7932, July 1998 Biochemistry The tyrosine kinase regulator Cbl enhances the ubiquitination and degradation of the platelet-derived growth factor receptor a SACHIKO MIYAKE*, MARK L. LUPHER,JR.*, BRIAN DRUKER†, AND HAMID BAND*‡ *Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and †Division of Heamatology and Medical Oncology, Oregon Health Sciences University, Portland, OR 97201 Communicated by K. Frank Austen, Harvard Medical School, Boston, MA, May 6, 1998 (received for review March 10, 1998) ABSTRACT The Cbl protooncogene product has emerged rapidly phosphorylated on tyrosine upon stimulation of cells as a negative regulator of receptor and nonreceptor tyrosine through a number of receptor tyrosine kinases as well as kinases. We recently demonstrated that oncogenic Cbl mu- receptors linked to nonreceptor tyrosine kinases (5–8). Nota- tants upregulate the endogenous tyrosine kinase signaling bly, Cbl is either constitutively (e.g., Src-family kinases) or machinery when expressed in the NIH 3T3 cells, and identified inducibly (e.g., EGFR, PDGFRa,orSykyZAP70 family) the platelet-derived growth factor receptor-a (PDGFRa)as associated with tyrosine kinases themselves (5–8, 12–16). A one of the tyrosine kinases targeted by these oncogenes. These critical determinant of the inducible association of Cbl with findings suggested a role for the normal Cbl protein in tyrosine kinases was identified recently. A phosphotyrosine- negative regulation of the PDGFRa. However, the mechanism binding (PTB) domain within the N-terminal transforming of such negative regulation remained to be determined. Here region of Cbl, corresponding to sequences retained in the v-cbl we show that overexpression of the wild-type Cbl enhances the oncogene, interacts directly with a specific phosphopeptide ligand-induced ubiquitination of the PDGFRa. Concomi- motif on tyrosine kinases to mediate a phosphorylation- tantly, the PDGFRa in Cbl-overexpressing cells undergoes a dependent association between Cbl and protein tyrosine ki- faster ligand-induced degradation compared with that in the nases (14, 15). In certain cases, such as the EGFR and control cells. These results identify a role for Cbl in the PDGFRa, a second indirect mechanism of association is regulation of ligand-induced ubiquitination and degradation provided by the Grb2 adaptor protein, which binds to the of receptor tyrosine kinases and suggest one potential mech- proline-rich region of Cbl via its SH3 domains and to auto- anism for evolutionarily conserved negative regulatory influ- phosphorylated receptors via its SH2 domain (10, 17, 18). ence of Cbl on tyrosine kinases. Notably, the N-terminal PTB domain-containing region (Cbl-N) and an adjacent RING finger domain are the only two Tyrosine kinases provide a universal mode of signal transmis- elements of Cbl highly conserved during evolution (5–8). sion in response to extracellular cues that regulate cell prolif- Indeed, Drosophila Cbl is exclusively composed of these two eration and differentiation. Uncontrolled activation of ty- regions, but nevertheless interacts with the EGFR (10). rosine kinases is implicated in proliferation of cancerous cells, The first evidence for the role of Cbl in negative regulation and their deficiencies result in pathological conditions such as of tyrosine kinase signaling pathways was provided by studies developmental abnormalities and immunodeficiencies (1). of vulval development in C. elegans, a process that requires Tight regulation of tyrosine kinase cascades is therefore LET-23 receptor tyrosine kinase, a homologue of the mam- critical to elicit an appropriate type and level of response to malian EGFR (9). A gene identified as a negative regulator of external stimuli. signaling downstream of the LET-23 receptor, suppressor of Negative regulation of tyrosine kinase-mediated signaling is lineage defect 1 (sli-1), encoded a Cbl homologue, SLI-1. achieved through a number of distinct biochemical mecha- Importantly, loss-of-function mutations identified in SLI-1 nisms. Cellular phosphotyrosine phosphatases provide one corresponded to either premature termination or a missense mechanism to reverse tyrosine kinase signals by removal of the mutation in the conserved Cbl-N homology region. In the phosphate moiety from phosphotyrosine (2). Another mech- Drosophila eye, a functional EGFR homologue is critical for anism to regulate tyrosine kinase signaling is through the the development of the R7 photoreceptor cell and functions removal of receptors, such as epidermal growth factor receptor through a Rasymitogen-activated protein kinase pathway anal- (EGFR) and platelet-derived growth factor receptor ogous to the mammalian EGFR. The recently cloned Dro- (PDGFR), from the cell surface via ligand-induced clathrin sophila Cbl homologue (D-Cbl) was shown to bind to Dro- coat-mediated endocytosis (3, 4). A potent and evolutionarily sophila and mammalian EGFRs and, when expressed as a conserved mechanism to regulate enzyme functions is through transgene under control of a sevenless enhancer, induced the direct interactions with regulatory proteins. However, tyrosine severe reduction in the development of the R7 photoreceptor, kinase regulatory proteins have not been previously identified. providing further evidence for a negative regulatory role of Cbl Recent biochemical studies in mammalian cells (5–8) and in receptor tyrosine kinase signaling (10). independent genetic studies in Caenorhabditis elegans and In mammalian systems, Cbl overexpression in NIH 3T3 cells Drosophila have identified the protooncogene product Cbl as correlated with decreased autophosphorylation of the EGFR a negative regulator of tyrosine kinases (9, 10). and lower JAK-STAT phosphorylation; conversely, higher Cbl was first identified as the cellular homologue of a viral EGFR autophosphorylation was observed in cells expressing oncogene v-cbl, which induces pre-B lymphomas and myeloid lower levels of Cbl as a consequence of antisense Cbl trans- leukemias in mice (11). A large number of studies have now fection (19). Cbl overexpression in the rat basophilic leukemia established that Cbl, a cytoplasmic 120 kDa polypeptide, is Abbreviations: PDGF, platelet-derived growth factor; PDGFR, plate- The publication costs of this article were defrayed in part by page charge let-derived growth factor receptor; EGFR, epidermal growth factor receptor; PTB, phosphotyrosine binding; HRP, horseradish peroxi- payment. This article must therefore be hereby marked ‘‘advertisement’’ in dase; HA, hemagglutinin. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: Smith Building, © 1998 by The National Academy of Sciences 0027-8424y98y957927-6$2.00y0 Room 538C, 75 Francis Street, Boston, MA 02115. e-mail:hband@ PNAS is available online at http:yywww.pnas.org. rics.bwh.harvard.edu. 7927 7928 Biochemistry: Miyake et al. Proc. Natl. Acad. Sci. USA 95 (1998) cell line RBL-2H3 was shown to decrease the autophosphor- containing 20 mM Hepes buffer solution (pH 7.35) and then ylation and kinase activity of Syk that is induced on Fc«R1 incubated in the same buffer with sulfo-N-hydroxysulfosuccin- stimulation, and mast cell degranulation was severely impaired imide (NHS)-biotin (Pierce) for 15 min at 4°C. After washing, (20). The in vivo negative regulatory phosphorylation site the cells were incubated in a-MEM containing 1 mgyml BSA pY292 was identified as the Cbl PTB domain-binding site on (tissue culture grade; Sigma) with or without PDGF for 15 min ZAP-70 (16). NIH 3T3 cells expressing oncogenic forms of Cbl at 4°C. The cells were washed, incubated at 37°C in a-MEM for revealed a hyperphosphorylation of the PDGF receptor a the indicated times, and lysates were prepared as described (PDGFRa) and an up-regulation of signaling downstream of above. Anti-PDGFRa immunoprecipitates of these lysates this receptor (14). Similarly, NIH 3T3 cells expressing onco- were blotted with avidin-HRP conjugate (Vector Laborato- genic forms of Cbl showed increased autophosphorylation and ries) followed by enhanced chemiluminescence, as described kinase activity of a transfected human EGFR, both under above. Densitometry was carried out by using the Hewlett– serum-starved and EGF-stimulated conditions (21). Appar- Packard ScanJet 4C and Scion Images for Windows software. ently, the effect of oncogenic mutants of Cbl on PDGFRa and Densitometric data were expressed as arbitrary units. EGFR reflects a reversal of the negative regulatory role of wild-type Cbl. RESULTS AND DISCUSSION Together, these studies strongly support a role for Cbl as a negative regulator of diverse tyrosine kinases. However, the Cbl Enhances the Ligand-Induced Ubiquitination of mechanism whereby Cbl negatively regulates tyrosine kinases PDGFRa. Our earlier studies demonstrated that the PDGFRa has not been elucidated. Here we demonstrate that Cbl was hyperactive in NIH 3T3 cells expressing various oncogenic promotes ubiquitination and ligand-induced degradation of mutants of Cbl when compared with cells expressing wild-type the receptor tyrosine kinase PDGFRa, suggesting one mech- Cbl, and led us to suggest that normal Cbl functions as a anism for the negative regulatory action of Cbl on tyrosine negative regulator of PDGFRa signaling (14). In these earlier kinases. studies, we noted that the kinetics