title: Biochemical basis for the requirement of kinase activity for Cbl-dependent ubiquitinylation and degradation of a target tyrosine kinase.
abstract: Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.
authors.affiliation: Division of Molecular Oncology, Department of Medicine Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60201, USA.
authors.forename: Amiya K
authors.initials: AK
authors.lastname: Ghosh
authors.forename: Alagarsamy L
authors.initials: AL
authors.lastname: Reddi
authors.forename: Navin L
authors.initials: NL
authors.lastname: Rao
authors.forename: Lei
authors.initials: L
authors.lastname: Duan
authors.forename: Vimla
authors.initials: V
authors.lastname: Band
authors.forename: Hamid
authors.initials: H
authors.lastname: Band
mesh_terms: D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008968:Molecular Conformation; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases