Katarzyna Kowanetz

Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors

Cbl is a multi-adaptor protein involved in ligand-induced downregulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction. Here we demonstrate that Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K; ref. 6) and endophilins (regulatory components of clathrin-coated vesicles) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 was constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl was increased on EGF stimulation. Inhibition of these interactions was sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases.

Regulation of ubiquitin-binding proteins by monoubiquitination

Proteins containing ubiquitin-binding domains (UBDs) interact with ubiquitinated targets and regulate diverse biological processes, including endocytosis, signal transduction, transcription and DNA repair. Many of the UBD-containing proteins are also themselves monoubiquitinated, but the functional role and the mechanisms that underlie this modification are less well understood. Here, we demonstrate that monoubiquitination of the endocytic proteins Sts1, Sts2, Eps15 and Hrs results in intramolecular interactions between ubiquitin and their UBDs, thereby preventing them from binding in trans to ubiquitinated targets. Permanent monoubiquitination of these proteins, mimicked by the fusion of ubiquitin to their carboxyl termini, impairs their ability to regulate trafficking of ubiquitinated receptors. Moreover, we mapped the in vivo monoubiquitination site in Sts2 and demonstrated that its mutation enhances the Sts2-mediated effects of epidermal-growth-factor-receptor downregulation. We propose that monoubiquitination of ubiquitin-binding proteins inhibits their capacity to bind to and control the functions of ubiquitinated targets in vivo.

CIN85 Participates in Cbl-b-mediated Down-regulation of Receptor Tyrosine Kinases

The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.

TGFβ induces SIK to negatively regulate type I receptor kinase signaling

Signal transduction by transforming growth factor β (TGFβ) coordinates physiological responses in diverse cell types. TGFβ signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFβ signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFβ/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFβ. We thus identify in SIK a negative regulator that controls TGFβ receptor turnover and physiological signaling.

Dab2 links CIN85 with clathrin-mediated receptor internalization

CIN85 is a multidomain scaffold protein involved in downregulation of receptor tyrosine kinases. Here we show that disabled‐2 (Dab2), an endocytic adaptor molecule implicated in clathrin‐coat assembly, associates with CIN85 in mammalian cells. All three SH3 domains of CIN85 were able to bind to the PKPAPR peptide in the carboxyl‐terminal part of Dab2, possibly enabling CIN85 to simultaneously interact with multiple Dab2 molecules. CIN85 association with Dab2 is essential for its recruitment to clathrin coat and appears to be modulated by growth factor stimulation. Dab2 and clathrin dissociated from CIN85 following growth factor treatment, enabling other molecules, such as Cbl, to bind to CIN85. Taken together, our data indicate a dynamic interplay between CIN85 and its effectors during endocytosis of receptor tyrosine kinases.

Transcriptional induction of salt-inducible kinase 1 by transforming growth factor β leads to negative regulation of type I receptor signaling in cooperation with the Smurf2 ubiquitin ligase

Background: The control of TGFβ signaling depends on many not well understood regulators. Results: TGFβ transcriptionally induces SIK1, which cooperates with the ubiquitin ligase Smurf2 to negatively regulate the signaling output. Conclusion: Transcriptional induction of SIK1 controls TGFβ signaling together with Smurf2 and Smad7. Significance: The molecular interplay between SIK1 and Smurf2 provides new means for controlling TGFβ signaling. Transforming growth factor β (TGFβ) regulates many physiological processes and requires control mechanisms to safeguard proper and timely action. We have previously described how negative regulation of TGFβ signaling is controlled by the serine/threonine kinase salt-inducible kinase 1 (SIK1). SIK1 forms complexes with the TGFβ type I receptor and with the inhibitory Smad7 and down-regulates the type I receptor. We now demonstrate that TGFβ induces SIK1 levels via a direct transcriptional mechanism that implicates the Smad proteins, and we have mapped a putative enhancer element on the SIK1 gene. We provide evidence that the ubiquitin ligase Smurf2 forms complexes and functionally cooperates with SIK1. Both the kinase activity of SIK1 and the ubiquitin ligase activity of Smurf2 are important for proper type I receptor turnover. We also show that knockdown of endogenous SIK1 and Smurf2 enhances physiological signaling by TGFβ that leads to epithelial growth arrest. In conclusion, TGFβ induces expression of Smad7, Smurf2, and SIK1, the products of which physically and functionally interlink to control the activity of this pathway.

erratum: Cbl–CIN85–endophilin complex mediates ligand-induced downregulation of EGF receptors

This corrects the article DOI: 416183a