Abstract
Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.
Highlights
The E3 ubiquitin ligase, c-Cbl [20, 21], recognizes phosphorylated tyrosine residues present on activated Receptor tyrosine kinase (RTK) via the tyrosine kinase binding (TKB) module. It recruits ubiquitin-conjugating enzymes or ubiquitin-carrier enzymes by the RING finger domain and catalyzes RTK ubiquitination, which leads to receptor internalization and degradation. c-Cbl is primarily expressed in hematopoietic cells [23, 24] and in macrophages, c-Cbl-deficiency results in compromised CSF-1 receptor (CSF-1R) ubiquitination, endocytosis, and degradation [25]. c-Cbl either binds CSF-1R directly [26], or through other molecules such as Grb2, PI3K [27], or Src-like adaptor protein 2 (SLAP-2), a hematopoietic adaptor protein which interacts with the CSF-1R and c-Cbl and plays a role in c-Cbl-dependent down-regulation of CSF-1R signaling [28]
We have studied Colony-stimulating factor-1 (CSF-1)-induced CSF-1R tyrosine phosphorylation and ubiquitination and mapped the structural element participating in receptor dimerization
Our results indicate that CSF-1 binding leads to CSF-1R dimerization and phosphorylation of Tyr-559 in the juxtamembrane domain (JMD) of CSF-1R which, apart from relieving autoinhibition of receptor kinase activity, activates a Src family kinases (SFKs)/c-Cbl pathway that mediates CSF-1R ubiquitination
Summary
We demonstrate a critical role of the CSF-1R JMD and the E3 ubiquitin ligase c-Cbl in mediating an interaction between the major kinase domains of the receptor that amplifies CSF-1R tyrosine phosphorylation. To investigate whether CSF-1-induced receptor interchain disulfide bonding reflects CSF-1R activation, we expressed the kinase-dead mutant K614A CSF-1R and tyrosine phosphorylation-defective mutant YEF CSF-1R in MϪ/Ϫ cells.
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