Load-induced regulation of tendon homeostasis by SPARC, a genetic predisposition factor for tendon and ligament injuries.

Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc -/- mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc -/- mice. Tendon hypoplasia was attenuated in Sparc -/- mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc -/- three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc -/- tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.

3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon-derived stem cells through a PI3K/AKT signaling pathway.

The tendon is a mechanosensitive tissue, but little is known about how mechanical stimulation selectively signals tenogenic differentiation and neo-tendon formation. In this study, we compared the impact of uniaxial and biaxial mechanical loading on tendon-derived stem cells (TDSCs). Our data show that there are variations in cell signaling and cell differentiation of mouse TDSCs in response to uniaxial and biaxial loading in monolayer culture. Whereas uniaxial loading induced TDSCs toward tenogenic and osteogenic differentiation, biaxial loading induced osteogenic, adipogenic, and chondrogenic differentiation of TDSCs. Furthermore, by applying uniaxial loading on 3-dimensional (3D) TDSC constructs, tenogenic-specific differentiation and neo-tendon formation were observed, results that were replicated in human TDSCs. We also showed that uniaxial loading induced PKB (AKT) phosphorylation (pAKT), whereas biaxial loading induced pERK. Most importantly, we found that inhibition of the PI3K/AKT signaling pathway could attenuate tenogenic differentiation and tendon formation in 3D TDSC constructs subjected to uniaxial loading. Taken together, our study highlights the importance of appropriate mechanobiological stimulation in 3D cell niches on tendon-like tissue formation and demonstrates that uniaxial mechanical loading plays an essential role in tenogenic differentiation and tendon formation by activating the PI3K/AKT signaling pathway.-Wang, T., Thien, C., Wang, C., Ni, M., Gao, J., Wang, A., Jiang, Q., Tuan, R. S., Zheng, Q., Zheng, M. H. 3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon-derived stem cells through a PI3K/AKT signaling pathway.

Cyclic mechanical stimulation rescues achilles tendon from degeneration in a bioreactor system.

Physiotherapy is one of the effective treatments for tendinopathy, whereby symptoms are relieved by changing the biomechanical environment of the pathological tendon. However, the underlying mechanism remains unclear. In this study, we first established a model of progressive tendinopathy-like degeneration in the rabbit Achilles. Following ex vivo loading deprivation culture in a bioreactor system for 6 and 12 days, tendons exhibited progressive degenerative changes, abnormal collagen type III production, increased cell apoptosis, and weakened mechanical properties. When intervention was applied at day 7 for another 6 days by using cyclic tensile mechanical stimulation (6% strain, 0.25 Hz, 8 h/day) in a bioreactor, the pathological changes and mechanical properties were almost restored to levels seen in healthy tendon. Our results indicated that a proper biomechanical environment was able to rescue early-stage pathological changes by increased collagen type I production, decreased collagen degradation and cell apoptosis. The ex vivo model developed in this study allows systematic study on the effect of mechanical stimulation on tendon biology.

Loss of c-Cbl E3 ubiquitin ligase activity enhances the development of myeloid leukemia in FLT3-ITD mutant mice.

Mutations in the Fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase (RTK) occur frequently in acute myeloid leukemia (AML), with the most common involving internal tandem duplication (ITD) within the juxtamembrane domain. Fms-like tyrosine kinase 3-ITD mutations result in a mislocalized and constitutively activated receptor, which aberrantly phosphorylates signal transducer and activator of transcription 5 (STAT5) and upregulates the expression of its target genes. c-Cbl is an E3 ubiquitin ligase that negatively regulates RTKs, including FLT3, but whether it can downregulate mislocalized FLT3-ITD remains to be resolved. To help clarify this, we combined a FLT3-ITD mutation with a loss-of-function mutation in the RING finger domain of c-Cbl that abolishes its E3 ligase activity. Mice transplanted with hematopoietic stem cells expressing both mutations rapidly develop myeloid leukemia, indicating strong cooperation between the two. Although the c-Cbl mutation was shown to cause hyperactivation of another RTK, c-Kit, it had no effect on enhancing FLT3-ITD protein levels or STAT5 activation. This indicates that c-Cbl does not downregulate FLT3-ITD and that the leukemia is driven by independent pathways involving FLT3-ITD's activation of STAT5 and mutant c-Cbl's activation of other RTKs, such as c-Kit. This study highlights the importance of c-Cbl's negative regulation of wild-type RTKs in suppressing FLT3-ITD-driven myeloid leukemia.

Dasatinib targets B-lineage cells but does not provide an effective therapy for myeloproliferative disease in c-Cbl RING finger mutant mice.

This study aimed to determine whether the multi-kinase inhibitor dasatinib would provide an effective therapy for myeloproliferative diseases (MPDs) involving c-Cbl mutations. These mutations, which occur in the RING finger and linker domains, abolish the ability of c-Cbl to function as an E3 ubiquitin ligase and downregulate activated protein tyrosine kinases. Here we analyzed the effects of dasatinib in a c-Cbl RING finger mutant mouse that develops an MPD with a phenotype similar to the human MPDs. The mice are characterized by enhanced tyrosine kinase signaling resulting in an expansion of hematopoietic stem cells, multipotent progenitors and cells within the myeloid lineage. Since c-Cbl is a negative regulator of c-Kit and Src signaling we reasoned that dasatinib, which targets these kinases, would be an effective therapy. Furthermore, two recent studies showed dasatinib to be effective in inhibiting the in vitro growth of cells from leukemia patients with c-Cbl RING finger and linker domain mutations. Surprisingly we found that dasatinib did not provide an effective therapy for c-Cbl RING finger mutant mice since it did not suppress any of the hematopoietic lineages that promote MPD development. Thus we conclude that dasatinib may not be an appropriate therapy for leukemia patients with c-Cbl mutations. We did however find that dasatinib caused a marked reduction of pre-B cells and immature B cells which correlated with a loss of Src activity. This study is therefore the first to provide a detailed characterization of in vivo effects of dasatinib in a hematopoietic disorder that is driven by protein tyrosine kinases other than BCR-ABL.

Flt3 inhibitor AC220 is a potent therapy in a mouse model of myeloproliferative disease driven by enhanced wild-type Flt3 signaling.

High levels of expression of wild-type Flt3 characterize many hematopoietic proliferative diseases and neoplasms, providing a potential therapeutic target. Using the c-Cbl RING finger mutant mouse as a model of a myeloproliferative disease (MPD) driven by wild-type Flt3, in the present study, we show that treatment with the Flt3 kinase inhibitor AC220 blocks MPD development by targeting Flt3(+) multipotent progenitors (MPPs). We found that daily administration of AC220 caused a marked reduction in Flt3 expression, induction of quiescence, and a significant loss of MPPs within 4 days. Unexpectedly, a robust Flt3 ligand-associated proliferative recovery response soon followed, preventing further loss of MPPs. However, continued AC220 treatment limited MPP recovery and maintained reduced, steady-state levels of cycling MPPs that express low levels of Flt3. Therefore, a finely tuned balance between the opposing forces of AC220 and Flt3 ligand production was established; whereas the Flt3 ligand blunted the inhibitory effects of AC220, the disease was held in remission for as long as therapy was continued. The net effect is a potent therapy indicating that patients with c-Cbl mutations, or those with similarly enhanced Flt3 signaling, may respond well to AC220 even after the induction of high levels of Flt3 ligand.

Incidence of c-Cbl mutations in human acute myeloid leukaemias in an Australian patient cohort.

AIM: The aim of this study was to investigate the incidence and characteristics of c-Cbl mutations in acute myeloid leukaemias (AMLs) from an Australian patient cohort. Two initial studies examining c-Cbl mutations in AML, one from Germany and one from the US, found vastly different incidences of mutations (0.6% compared to 33%, respectively). Therefore, it was important to determine the incidence and characteristics of c-Cbl mutations in a cohort of Australian AML patients. METHODS: Ninety patients with AML were investigated. The open reading frame between exons 4 and 11 of the c-Cbl gene was analysed by reverse-transcription polymerase chain reaction (RT-PCR), nested PCR and DNA sequencing. RESULTS: We found four AML samples (4/90; 4.44%) with distinct c-Cbl deletions involving exons 6 to 9. Sample 10 [AML with t(8;21)] showed two deletions [c.870-1007del] and [c.1106-1228del]. Sample 81 (AML with minimal differentiation) showed a large deletion [c.1008-1431del] causing a frameshift and a premature stop codon. Sample 82 (AML without maturation) showed two deletions [c.928-1307del] and [c.1385-1431del] also causing a frameshift and a premature stop codon. Sample 84 (AML with myelodysplasia related changes) showed a large deletion [c.964-1380del]. CONCLUSION: Although our data indicate that c-Cbl deletions are not common in AML in the Australian population, they do raise the possibility that c-Cbl mutations might contribute to the pathogenesis of these AML cases.

Essential role of E3 ubiquitin ligase activity in Cbl-b-regulated T cell functions.

E3 ubiquitin ligases have been placed among the essential molecules involved in the regulation of T cell functions and T cell tolerance. However, it has never been experimentally proven in vivo whether these functions indeed depend on the catalytic E3 ligase activity. The Casitas B-cell lymphoma (Cbl) family protein Cbl-b was the first E3 ubiquitin ligase directly implicated in the activation and tolerance of the peripheral T cell. In this study, we report that selective genetic inactivation of Cbl-b E3 ligase activity phenocopies the T cell responses observed when total Cbl-b is ablated, resulting in T cell hyperactivation, spontaneous autoimmunity, and impaired induction of T cell anergy in vivo. Moreover, mice carrying a Cbl-b E3 ligase-defective mutation spontaneously reject tumor cells that express human papilloma virus Ags. These data demonstrate for the first time, to our knowledge, that the catalytic function of an E3 ligase, Cbl-b, is essential for negative regulation of T cells in vivo. Thus, modulation of the E3 ligase activity of Cbl-b might be a novel modality to control T cell immunity in vaccination, cancer biology, or autoimmunity.

Visualizing the role of Cbl-b in control of islet-reactive CD4 T cells and susceptibility to type 1 diabetes.

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4(+) T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4(+) T cells in prediabetic 3A9-TCR × insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4(+) cells or the numbers of islet-specific CD4(+) or CD4(+)Foxp3(+) T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-ß-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4(+) cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4(+) cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity.

Myeloid leukemia development in c-Cbl RING finger mutant mice is dependent on FLT3 signaling.

Although myeloid leukemias are primarily caused by leukemic stem cells, the molecular basis of their transformation remains largely unknown. Here, by analyzing mice with a mutation in the RING finger domain of c-Cbl, we show that the E3 ubiquitin ligase activity of c-Cbl is required to restrict myeloid leukemia development. These mice develop a myeloproliferative disease which progresses to leukemia and involves hematopoietic progenitors that exhibit augmented FLT3 signaling. Suppressing this signaling through matings with FLT3 ligand knockout mice prevents leukemia development. We also observe enhanced c-Kit, Akt and Erk activity, and deregulated expression of leukemia-associated transcription factors in hematopoietic progenitors. The characterization of these perturbations provides direction for therapeutics that may aid the treatment of patients with c-Cbl mutations.

c-Cbl promotes T cell receptor-induced thymocyte apoptosis by activating the phosphatidylinositol 3-kinase/Akt pathway.

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

Unaltered negative selection and Treg development of self-reactive thymocytes in TCR transgenic Fyn-deficient mice.

The tyrosine kinase Fyn has been implicated as playing an important role in the generation of both stimulatory and inhibitory signaling events induced by TCR engagement. To assess the role of Fyn for antigen-driven negative selection and Treg development, which are both dependent on the strength and nature of TCR signaling, we generated mice that co-express the transgenes for OVA and the OT-II TCR, which recognizes a peptide from OVA. In mice expressing both transgenes, negative selection, Treg development in the thymus, and the number of Treg in the periphery were each unaffected by ablation of Fyn. Moreover, fyn(-/-) Treg were functional, as assessed in vitro. We further tested the role of Fyn for the adaptor function of c-Cbl, using mice containing a point mutation in c-Cbl that abolishes its E3 ubiquitin ligase function but maintains its adaptor function. The functional and signaling properties of this mutant c-Cbl were unaltered in fyn(-/-) thymocytes. Combined, these data indicate that Fyn was not required for the induction of central tolerance by negative selection, the adaptor protein role of c-Cbl, or the normal development and function of Treg.

A TNF- and c-Cbl-dependent FLIP(S)-degradation pathway and its function in Mycobacterium tuberculosis-induced macrophage apoptosis.

Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl. This pathway led to phosphorylation of FLIP(S), which facilitated its interaction with the E3 ubiquitin ligase c-Cbl. This interaction triggered proteasomal degradation of FLIP(S), which promoted activation of caspase-8 and apoptosis. Our findings identify a previously unappreciated signaling pathway needed for Mycobacterium tuberculosis-triggered macrophage cell death.

The E3 ubiquitin ligase c-Cbl restricts development and functions of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are multipotent progenitors that give rise to all types of blood cells. In the present study, we document that HSC development and functions are negatively regulated by the E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma). HSCs of c-Cbl(-/-) mice exhibit augmented pool size, hyperproliferation, greater competence, and enhanced long-term repopulating capacity. Our mechanistic studies identified that c-Cbl(-/-) HSCs are hyperresponsive to thrombopoietin (TPO) and display elevated levels of STAT5 phosphorylation, thus leading to increased c-Myc expression. In essence, our data unequivocally identify c-Cbl as a novel negative regulator of developmental and functional properties of HSCs.

The Cbl-b RING finger domain has a limited role in regulating inflammatory cytokine production by IgE-activated mast cells.

The RING finger type E3 ubiquitin ligase, Cbl-b, is abundantly expressed in bone marrow-derived mast cells (BMMCs) and functions as a potent negative regulator of signalling responses from the high-affinity IgE receptor (FcvarepsilonRI). To determine the contribution of Cbl-b E3 ligase activity we generated knockin mice with a loss-of-function mutation in the RING finger domain. We find the mice to be healthy and, unlike equivalent c-Cbl RING finger mutant mice, produce homozygous offspring at the expected frequency. Comparative analyses of BMMCs from Cbl-b knockout and Cbl-b RING finger mutant mice revealed that both showed similarly enhanced FcvarepsilonRI signalling compared to wild-type cells for most parameters examined. A notable exception was a markedly higher level of activation of IkappaB kinase (IKK) in Cbl-b knockout BMMC compared to RING finger mutant-derived cells. In addition BMMCs from the Cbl-b RING finger mutant did not retard FcvarepsilonRI internalization to the extent observed for knockout cells. Most striking however was the finding that RING finger mutant mast cells do not produce the very high levels of TNF-alpha, IL-6, and MCP-1 evident in Cbl-b knockout cultures following FcvarepsilonRI activation. Thus the ability of Cbl-b to function as a negative regulator of FcvarepsilonRI signalling that promotes inflammatory cytokine production is largely independent of the RING finger domain.

Genetic ablation of the c-Cbl ubiquitin ligase domain results in increased energy expenditure and improved insulin action.

Casitas b-lineage lymphoma (c-Cbl) is a multiadaptor protein with E3-ubiquitin ligase activity residing within its RING finger domain. We have previously reported that c-Cbl-deficient mice exhibit elevated energy expenditure, reduced adiposity, and improved insulin action. In this study, we examined mice expressing c-Cbl protein with a loss-of-function mutation within the RING finger domain (c-Cbl(A/-) mice). Compared with control animals, c-Cbl(A/-) mice display a phenotype that includes reduced adiposity, despite greater food intake; reduced circulating insulin, leptin, and triglyceride levels; and improved glucose tolerance. c-Cbl(A/-) mice also display elevated oxygen consumption (13%) and are protected against high-fat diet-induced obesity and insulin resistance. Unlike c-Cbl(A/-) mice, mice expressing a mutant c-Cbl with the phosphatidylinositol (PI) 3-kinase binding domain ablated (c-Cbl(F/F) mice) exhibited an insulin sensitivity, body composition, and energy expenditure similar to that of wild-type animals. These results indicate that c-Cbl ubiquitin ligase activity, but not c-Cbl-dependent activation of PI 3-kinase, plays a key role in the regulation of whole-body energy metabolism.

Cbl-b is a negative regulator of inflammatory cytokines produced by IgE-activated mast cells.

c-Cbl and Cbl-b E3 ubiquitin ligases are abundantly expressed in hemopoietic cells where they negatively regulate the activity and levels of many cell surface receptors and associated signaling molecules. By comparing bone marrow-derived mast cells from c-Cbl and Cbl-b-deficient mice it has recently been shown that Cbl-b is the dominant family member for negatively regulating signaling responses from high-affinity IgE receptors. In this study, we suggest that a possible reason for the greater enhancement of IgE receptor signaling in Cbl-b-deficient mice is the relatively higher levels of Cbl-b protein over c-Cbl in mast cells compared with other hemopoietic cells. We also directly compare mast cells from c-Cbl and Cbl-b-deficient mice and find that loss of Cbl-b, but not c-Cbl, increases cell growth, retards receptor internalization, and causes the sustained tyrosine phosphorylation of Syk and its substrates. However, loss of Cbl-b does not enhance the activation of ERK or Akt, nor does it promote a greater calcium response. Furthermore, loss of Cbl-b or c-Cbl does not increase levels of the Syk or Lyn protein tyrosine kinases. Most notable, however, is the extremely large increase in the production of proinflammatory cytokines TNF-alpha, IL-6, and MCP-1 by Cbl-b(-/-) mast cells compared with levels produced by c-Cbl(-/-) or wild-type cells. This marked induction, which appears to be restricted to these three cytokines, is dependent on IgE receptor activation and correlates with enhanced IkappaB kinase phosphorylation. Thus, Cbl-b functions as a potent negative regulator of cytokines that promote allergic and inflammatory reactions.

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion.

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.

c-Cbl and Cbl-b ubiquitin ligases: substrate diversity and the negative regulation of signalling responses.

The activation of signalling pathways by ligand engagement with transmembrane receptors is responsible for determining many aspects of cellular function and fate. While these outcomes are initially determined by the nature of the ligand and its receptor, it is also essential that intracellular enzymes, adaptor proteins and transcription factors are correctly assembled to convey the intended response. In recent years, it has become evident that proteins that regulate the amplitude and duration of these signalling responses are also critical in determining the function and fate of cells. Of these, the Cbl family of E3 ubiquitin ligases and adaptor proteins has emerged as key negative regulators of signals from many types of cell-surface receptors. The array of receptors and downstream signalling proteins that are regulated by Cbl proteins is diverse; however, in most cases, the receptors have a common link in that they either possess a tyrosine kinase domain or they form associations with cytoplasmic PTKs (protein tyrosine kinases). Thus Cbl proteins become involved in signalling responses at a time when PTKs are first activated and therefore provide an initial line of defence to ensure that signalling responses proceed at the desired intensity and duration.

Negative regulation of PTK signalling by Cbl proteins.

Signalling pathways that are activated by ligands binding to cell surface receptors are responsible for determining many aspects of cellular function and fate. Although this outcome is primarily determined by the nature of the ligand and its receptor, it is also essential that the array of intracellular enzymes, adaptor proteins and transcription factors are correctly assembled to convey the intended response. In recent years it has become apparent that proteins which regulate the amplitude and duration of these responses can also affect cell function and fate. The Cbl family of E3 ubiquitin ligases and adaptor proteins have now emerged as key negative regulators of signals from many surface receptors. Although the array of these receptors is diverse, they have a common link in that they either possess a tyrosine kinase domain or they form associations with cytoplasmic protein tyrosine kinases (PTKs). Thus Cbl proteins become involved in signaling responses at a time when PTKs are first activated and therefore provide an initial line of defense to ensure signaling responses proceed at the desired intensity and kinetics.