Ubiquitination and degradation of the hominoid- specific oncoprotein TBC1D3 Is mediated by CUL7 E3 ligase

Expression of the hominoid-specific TBC1D3 oncoprotein enhances growth factor receptor signaling and subsequently promotes cellular proliferation and survival. Here we report that TBC1D3 is degraded in response to growth factor signaling, suggesting that TBC1D3 expression is regulated by a growth factor-driven negative feedback loop. To gain a better understanding of how TBC1D3 is regulated, we studied the effects of growth factor receptor signaling on TBC1D3 post-translational processing and turnover. Using a yeast two-hybrid screen, we identified CUL7, the scaffolding subunit of the CUL7 E3 ligase complex, as a TBC1D3-interacting protein. We show that CUL7 E3 ligase ubiquitinates TBC1D3 in response to serum stimulation. Moreover, TBC1D3 recruits F-box 8 (Fbw8), the substrate recognition domain of CUL7 E3 ligase, in pull-down experiments and in an in vitro assay. Importantly, alkaline phosphatase treatment of TBC1D3 suppresses its ability to recruit Fbw8, indicating that TBC1D3 phosphorylation is critical for its ubiquitination and degradation. We conclude that serum- and growth factor-stimulated TBC1D3 ubiquitination and degradation are regulated by its interaction with CUL7-Fbw8

TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein

Trypanosoma cruzi : Mecanismos reguladores de la metaciclogénesis

En el intestino de su vector, insecto hematófago, Trypanosoma cruzi, se encuentra expuesto a una gran variedad de compuestos. Algunos de éstos inducen la diferenciación del parásito hacia su estadío de tripomastigote metacíclico, que es la forma infectante. Este proceso implica la activación de vias especificas de señalización. En esta Tesis investigamos la generación de moléculas liposolubles con actividad metaciclogénica y su mecanismo de acción. Demostramos que los ácidos grasos libres, juegan un rol importante en la metaciclogénesis, el que puede explicarse por la activación de la proteína kinasa C (PKC). La generación de estos ácidos grasos libres tiene lugar por efecto de fosfolipasas exógenas (del intestino del vector) y endógenas (del tripanosoma) actuando en forma concurrente. Estas fosfolipasas han sido aisladas y caracterizadas. Los ácidos grasos libres estimulan la generación de diacilglicerol, contribuyendo a explicar su efecto activador sobre la PKC. A este efecto, se suma la aparición de aumentos de calcio citoplásmico, por exposición a extractos intestinales, que ha sido demostrada anteriormente, con lo que se generan simultáneamente los dos segundos mensajeros (iones calcio y diacilglicerol), requeridos para la activación de la PKC. Estudios previos indicaron que la digestión de la hemoglobina genera péptidos metaciclogénicos que actúan a través de la proteina kinasa A (PKA) del parásito. Existen en consecuencia mecanismos redundantes, mediados por la PKC y la PKA, que aseguran la diferenciación celular en Trypanosoma cruzi. En ambos casos, péptidos de la hemoglobina y ácidos grasos libres, son productos de la digestión de la sangre. Un posible significado biológico de estas observaciones es que dichos productos desempeñen un rol coordinador entre la fisiología alimentaria del insecto y la aparición de las formas infectantes del parásito.Inside the intestinal medium of its blood sucking vector, Trypanosoma cruzi is exposed to large variety of compounds. Some of these induce the differentiation of the parasite to its infective metacyclic trypomastigote stage. This process implies the activation of specific signaling pathways. In this Thesis we investigate the generation of lipid soluble molecules with metacyclogenic activity and their mechanism of action. We show that free fatty acids play a key role in this process which can be explained through the activation of protein kinase C (PKC). The generation of those free fatty acids takes place through exogenous (intestinal) and endogenous (trypanosomal) phospholipases which act concurrently. These phospholipases have been characterized in this work. The free fatty acids stimulate the formation of diacylglycerol thus, accounting for the PKC-activating effects. Cytoplasmic calcium peaks have been demonstrated to take place upon stimulation with the intestinal extracts. Therefore, both second messengers, diacylglycerol and calcium; appear simultaneously as required for PKC activation. Previous work has shown that peptides derived from the digestion of hemoglobin, have metacyclogenic activity through the activation of protein kinase A (PKA) in the parasite. Thus, there are redundant mechanisms, involving PKC and PKA that ensure cell differentiation in Trypanosoma cruzi. In both cases, hemoglobin peptides and free fatty acids, the regulatory compounds, are blood digestion products. A possible biological meaning of these observations is that such products serve a coordinated role between the feeding physiology of the insect and the appearance of the infective forms of the parasite.Fil:Wainszelbaum, Marisa. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Free fatty acids induce cell differentiation to infective forms in Trypanosoma cruzi.

Intestinal extracts of Triatoma infestans induce cell differentiation of Trypanosoma cruzi epimastigotes into the infective metacyclic form. Part of this effect can be explained by the presence of haemoglobin fragments, which stimulate trypanosomal adenylate cyclase. In this work we examined the metacyclogenic activity of lipids present in this intestinal extract. We found that lipid extracts of the intestinal extract have significant stimulatory effects that reside with the free-fatty-acid fraction, especially oleic acid. These compounds stimulate de novo diacylglycerol formation and protein kinase C activity in the parasite. Moreover, metacyclogenesis is stimulated by phorbol esters and cell-permeant diacylglycerol, while protein kinase C down-regulation or incubation with inhibitors of this kinase abrogates this effect. These results indicate that free fatty acids are a novel signal, inducing metacyclogenesis, acting through a pathway involving diacylglycerol biosynthesis and protein kinase C activation

Model for the regulation of IRS-1 degradation by TBC1D3 expression.

<p>We propose that TBC1D3 suppresses the degradation of IRS-1 by regulating the phosphorylation of S6K at T389. In this model, mTOR phosphorylates S6K in response to insulin signaling. TBC1D3 interacts, directly or indirecly, with PP2A B56γ to enhance the dephosphorylation of S6K:T389 thereby reducing the S6K-dependent phosphorylation of IRS-1 at key sites which are required for IRS-1 ubiquitination and degradation.</p

TBC1D3 expression blocks IRS-1 degradation.

<p>IRS-1 degradation is delayed in cells expressing TBC1D3. DU145 cells transfected with myc-TBC1D3 or empty vector were serum-starved, and stimulated with insulin (10 nM) for the indicated times. Protein levels of IRS-1 were analyzed by Western blotting. (<i>Right panel</i>) Quantification data of IRS-1 normalized to GAPDH protein levels. The value of IRS-1 at time 0 was set at 1.0. The data are presented as means ± SD of three independent experiments.</p