The proliferation and differentiation of cells of many lineages are regulated by secreted proteins known as cytokines. Cytokines exert their biological effect through binding to cell-surface receptors that are associated with one or more members of the JAK family of cytoplasmic tyrosine kinases. Cytokine-induced receptor dimerization leads to the activation of JAKs, rapid tyrosine-phosphorylation of the cytoplasmic domains, and subsequent recruitment of various signalling proteins, including members of the STAT family of transcription factors, to the receptor complex. Using the yeast two-hybrid system, we have now isolated a new SH2-domain-containing protein, JAB, which is a JAK-binding protein that interacts with the Jak2 tyrosine-kinase JH1 domain. JAB is structurally related to CIS, a cytokine-inducible SH2 protein. Interaction of JAB with Jak1, Jak2 or Jak3 markedly reduces their tyrosine-kinase activity and suppresses the tyrosine-phosphorylation and activation of STATs. JAB and CIS appear to function as negative regulators in the JAK signalling pathway.

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N-and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, cCbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

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The protooncogene c-Cbl has recently emerged as an E3 ubiquitin ligase for activated receptor tyrosine kinases. We report here that c-Cbl also mediates the ubiquitination of another protooncogene, the non-receptor tyrosine kinase c-Src, as well as of itself. The c-Cbl-dependent ubiquitination of Src and c-Cbl requires c-Cbl's RING finger, Src kinase activity, and c-Cbl's tyrosine phosphorylation, probably on Tyr-371. In vitro, c-Cbl forms a stable complex with the ubiquitin-conjugating enzyme UbcH7, but active Src destabilizes this interaction. In contrast, Src inhibition stabilizes the c-Cbl⅐ UbcH7⅐Src complex. Finally, c-Cbl reduces v-Src protein levels and suppresses v-Src-induced STAT3 activation. Thus, in addition to mediating the ubiquitination of activated receptor tyrosine kinases, c-Cbl also acts as a ubiquitin ligase for the non-receptor tyrosine kinase Src, thereby down-regulating Src.The protein product of the c-cbl proto-oncogene is involved in several signaling pathways and exerts a negative regulatory function on receptor and non-receptor tyrosine kinases (1-7). Although the mechanism by which c-Cbl exerts this negative regulatory function remained unclear until recently, it is now apparent that in several instances c-Cbl works by promoting polyubiquitination of activated receptor protein-tyrosine kinases (RTKs) 1 (8 -16). The domains of c-Cbl that are most directly involved in the ubiquitination process are the RING domain (17) and a linker region that lies between the RING domain and the N-terminal phosphotyrosine-binding (PTB) domain (also termed tyrosine kinase binding or TKB domain 2 ) (16, 18 -20). Deletion of all or part of these residues, as in the oncogenic v-Cbl and 70Z-Cbl, abolishes the ability of c-Cbl to mediate ubiquitination of growth factor receptors, generating dominant negative versions of c-Cbl (17). Crystal structure analysis of the evolutionarily conserved N-terminal half of the c-Cbl molecule revealed that the PTB domain comprises three subdomains: a four-helix bundle, an EF-hand, and a divergent SH2 domain, all of which contribute residues necessary for the binding of phosphotyrosine-containing target proteins (21). The C-terminal-half of c-Cbl has a proline-rich region and several tyrosines, which are phosphorylated in response to various stimuli, as well as a leucine zipper domain that mediates the formation of c-Cbl homodimers (22). As yet, there is no evidence that the C-terminal domains play a role in the ubiquitinationrelated function of c-Cbl. The ubiquitin system, in which the recruitment of ubiquitinconjugating enzymes (E2) to specific proteins leads to their subsequent ubiquitination and degradation via the proteasome, selectively terminates the activity of many proteins in cells (23)(24)(25)(26)(27). This pathway generally involves the sequential action of three types of molecules: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). We and others (10,12,16) have independently demonstrated that c-Cbl can function a...