title: Phosphotyrosine binding domain-dependent upregulation of the platelet-derived growth factor receptor alpha signaling cascade by transforming mutants of Cbl: implications for Cbl's function and oncogenicity.
abstract: Recent studies have demonstrated that Cbl, the 120-kDa protein product of the c-cbl proto-oncogene, serves as a substrate of a number of receptor-coupled tyrosine kinases and forms complexes with SH3 and SH2 domain-containing proteins, pointing to its role in signal transduction. Based on genetic evidence that the Caenorhabditis elegans Cbl homolog, SLI-1, functions as a negative regulator of the LET-23 receptor tyrosine kinase and our demonstration that Cbl's evolutionarily conserved N-terminal transforming region (Cbl-N; residues 1 to 357) harbors a phosphotyrosine binding (PTB) domain that binds to activated ZAP-70 tyrosine kinase, we examined the possibility that oncogenic Cbl mutants may activate mitogenic signaling by deregulating cellular tyrosine kinase machinery. Here, we show that expression of Cbl-N and two other transforming Cbl mutants (CblY368 delta and Cbl366-382 delta or Cb170Z), but not wild-type Cbl, in NIH 3T3 fibroblasts leads to enhancement of endogenous tyrosine kinase signaling. We identified platelet-derived growth factor receptor alpha (PDGFR alpha) as one target of mutant Cbl-induced deregulation. In mutant Cbl transfectants, PDGFR alpha was hyperphosphorylated and constitutively complexed with a number of SH2 domain-containing proteins. PDGFR alpha hyperphosphorylation and enhanced proliferation of mutant Cbl-transfected NIH 3T3 cells were drastically reduced upon serum starvation, and PDGF-AA substituted for the maintenance of these traits. PDGF-AA stimulation of serum-starved Cbl transfectants induced the in vivo association of transfected Cbl proteins with PDGFR alpha. In vitro, Cbl-N directly bound to PDGFR alpha derived from PDGF-AA-stimulated cells but not to that from unstimulated cells, and this binding was abrogated by a point mutation (G306E) corresponding to a loss-of-function mutation in SLI-1. The Cbl-N/G306E mutant protein, which failed to induce enhanced growth and transformation of NIH 3T3 cells, also failed to induce hyperphosphorylation of PDGFR alpha. Altogether, these findings identify a novel mechanism of Cbl's physiological function and oncogenesis, involving its PTB domain-dependent direct interaction with cellular tyrosine kinases.
authors.affiliation: Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: D P
authors.initials: DP
authors.lastname: Bonita
authors.forename: S
authors.initials: S
authors.lastname: Miyake
authors.forename: M L
authors.initials: ML
authors.lastname: Lupher
authors.forename: W Y
authors.initials: WY
authors.lastname: Langdon
authors.forename: H
authors.initials: H
authors.lastname: Band
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D006801:Humans; D051379:Mice; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D044767:Ubiquitin-Protein Ligases; D015854:Up-Regulation / Q000502:physiology*; D018909:src Homology Domains
chemical_list: C000715470:MAS1 protein, human; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; C075278:platelet-derived growth factor A; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D020796:Receptor, Platelet-Derived Growth Factor alpha; D017479:Receptors, Platelet-Derived Growth Factor; C067419:CBL protein, human; C493849:Cbl protein, mouse