Asymmetric Dimethylation of Ribosomal S6 Kinase 2 Regulates Its Cellular Localisation and Pro-Survival Function.

Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated.

Dual regulation of fatty acid synthase (FASN) expression by O-GlcNAc transferase (OGT) and mTOR pathway in proliferating liver cancer cells.

Fatty acid synthase (FASN) participates in many fundamental biological processes, including energy storage and signal transduction, and is overexpressed in many cancer cells. We previously showed in a context of lipogenesis that FASN is protected from degradation by its interaction with O-GlcNAc transferase (OGT) in a nutrient-dependent manner. We and others also reported that OGT and O-GlcNAcylation up-regulate the PI3K/AKT/mTOR pathway that senses mitogenic signals and nutrient availability to drive cell cycle. Using biochemical and microscopy approaches, we show here that FASN co-localizes with OGT in the cytoplasm and, to a lesser extent, in the membrane fraction. This interaction occurs in a cell cycle-dependent manner, following the pattern of FASN expression. Moreover, we show that FASN expression depends on OGT upon serum stimulation. The level of FASN also correlates with the activation of the PI3K/AKT/mTOR pathway in hepatic cell lines, and in livers of obese mice and in a chronically activated insulin and mTOR signaling mouse model (PTEN-null mice). These results indicate that FASN is under a dual control of O-GlcNAcylation and mTOR pathways. In turn, blocking FASN with the small-molecule inhibitor C75 reduces both OGT and O-GlcNAcylation levels, and mTOR activation, highlighting a novel reciprocal regulation between these actors. In addition to the role of O-GlcNAcylation in tumorigenesis, our findings shed new light on how aberrant activity of FASN and mTOR signaling may promote the emergence of hepatic tumors.

Intravenous administration of scAAV9-Hexb normalizes lifespan and prevents pathology in Sandhoff disease mice.

Sandhoff disease (SD) is a rare inherited disorder caused by a deficiency of ß-hexosaminidase activity which is fatal because no effective treatment is available. A mouse model of Hexb deficiency reproduces the key pathognomonic features of SD patients with severe ubiquitous lysosomal dysfunction, GM2 accumulation, neuroinflammation and neurodegeneration, culminating in death at 4 months. Here, we show that a single intravenous neonatal administration of a self-complementary adeno-associated virus 9 vector (scAAV9) expressing the Hexb cDNA in SD mice is safe and sufficient to prevent disease development. Importantly, we demonstrate for the first time that this treatment results in a normal lifespan (over 700 days) and normalizes motor function assessed by a battery of behavioral tests, with scAAV9-treated SD mice being indistinguishable from wild-type littermates. Biochemical analyses in multiple tissues showed a significant increase in hexosaminidase A activity, which reached 10-15% of normal levels. AAV9 treatment was sufficient to prevent GM2 and GA2 storage almost completely in the cerebrum (less so in the cerebellum), as well as thalamic reactive gliosis and thalamocortical neuron loss in treated Hexb-/- mice. In summary, this study demonstrated a widespread protective effect throughout the entire CNS after a single intravenous administration of the scAAV9-Hexb vector to neonatal SD mice.

Genetics in biliary atresia.

PURPOSE OF REVIEW: Biliary atresia is a poorly understood deadly disease. Genetic predisposition factors are suspected albeit not firmly established. This review summarizes recent evidence of genetic alterations in biliary atresia. RECENT FINDINGS: Whole-genome association studies in biliary atresia patients identified four distinct predisposition loci with four different genes potentially involved in the disease occurrence. Variations in these genes were searched for, but none were found in patients with biliary atresia suggesting complex mechanisms. SUMMARY: Despite decades since its description and decades of intensive researches, cause of biliary atresia disease remains enigmatic. The inheritance of biliary atresia is not Mendelian. Genetic predisposition factor is one of the explored fields to explain biliary atresia pathogenicity. Biliary atresia has been associated with several inborn syndromes, chromosome anomalies, and gene polymorphisms in specific populations. Four predisposition loci encompassing genes relevant to the disease have been identified, but no pathogenic variations were found in biliary atresia patients. Few reported cases of isolated biliary atresia manifestation in the context of known genetic diseases suggest coincidental findings. Alternatives to classic genetic alterations are proposed to explain genetic predisposition in biliary atresia including noncoding and epigenetic factors. Biliary atresia is most likely related to complex traits making its genetic exploration challenging.

Role of PI3K, mTOR and Akt2 signalling in hepatic tumorigenesis via the control of PKM2 expression.

To sustain increased growth, rapidly proliferating cells, such as tumour cells, undergo metabolic adaptations. In recent years, the mechanisms of glycolysis activation as a key metabolic adaptation in proliferating cells became the topic of intense research. Although this phenomenon was described more than 50 years ago by Otto Warburg, the molecular mechanisms remained elusive. Only recently, it was demonstrated that the expression of specific glycolytic enzymes, namely PKM2 (pyruvate kinase M2) and HK2 (hexokinase 2), occurs simultaneously with the glycolytic addiction of cancer cells. The PI3K (phosphoinositide 3-kinase)/mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway is a central signalling hub co-ordinating the growth in response to growth factor signalling and nutrient availability. Not surprisingly, it is found to be activated in the majority of the tumour cells. In the present article, we discuss the requirement of different PI3K/mTOR downstream effectors for the metabolic adaptation in liver cancer cells driven by this signalling pathway. We provide evidence for a selective involvement of the mTOR target Akt2 in tumoral growth. In addition, PTEN (phosphatase and tensin homologue deleted on chromosome 10)-negative human hepatocellular carcinoma cell lines display an up-regulation of PKM2 expression in an Akt2-dependent manner, providing an advantage for cell proliferation and anchorage-independent growth. Our data have implications on the link between the metabolic action of insulin signal transduction and tumorigenesis, identifying Akt2 as a potential therapeutical target in liver malignancies depending on cancer genotype.

Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis.

Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1-Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1-Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1-Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat, a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat. Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis.

Design and Evaluation of Autophagy-Inducing Particles for the Treatment of Abnormal Lipid Accumulation.

Autophagy is a fundamental housekeeping process by which cells degrade their components to maintain homeostasis. Defects in autophagy have been associated with aging, neurodegeneration and metabolic diseases. Non-alcoholic fatty liver diseases (NAFLDs) are characterized by hepatic fat accumulation with or without inflammation. No treatment for NAFLDs is currently available, but autophagy induction has been proposed as a promising therapeutic strategy. Here, we aimed to design autophagy-inducing particles, using the autophagy-inducing peptide (Tat-Beclin), and achieve liver targeting in vivo, taking NAFLD as a model disease. Polylactic acid (PLA) particles were prepared by nanoprecipitation without any surfactant, followed by surface peptide adsorption. The ability of Tat-Beclin nanoparticles (NP T-B) to modulate autophagy and to decrease intracellular lipid was evaluated in vitro by LC3 immunoblot and using a cellular model of steatosis, respectively. The intracellular localization of particles was evaluated by transmission electron microscopy (TEM). Finally, biodistribution of fluorescent NP T-B was evaluated in vivo using tomography in normal and obese mice. The results showed that NP T-B induce autophagy with a long-lasting and enhanced effect compared to the soluble peptide, and at a ten times lower dose. Intracellular lipid also decreased in a cellular model of NAFLD after treatment with T-B and NP T-B under the same dose conditions. Ultrastructural studies revealed that NP T-B are internalized and located in endosomal, endolysosomal and autolysosomal compartments, while in healthy and obese mice, NP T-B could accumulate for several days in the liver. Given the beneficial effects of autophagy-inducing particles in vitro, and their capacity to target the liver of normal and obese mice, NP T-B could be a promising therapeutic tool for NAFLDs, warranting further in vivo investigation.

Class 3 phosphoinositide 3-kinase promotes hepatic glucocorticoid receptor stability and transcriptional activity.

AIM: Lipid kinase class 3 phosphoinositide 3-kinase (PI3K) and nuclear receptor transcription factor glucocorticoid receptor (GR) play essential physiological roles in metabolic adaptation to fasting by activating lysosomal degradation by autophagy and metabolic gene expression, yet their functional interaction is unknown. The requirement of class 3 PI3K for GR function was investigated in liver tissue. METHODS: Inactivation of class 3 PI3K was achieved through deletion of its essential regulatory subunit Vps15, by expressing Cre-recombinase in the livers of Vps15f/f mice. The response to both 24-h fasting and synthetic GR ligand, dexamethasone (DEX) was evaluated in control and mutant mice. Liver tissue was analysed by immunoblot, RT-qPCR, and LC-MS. RESULTS: Vps15 mutant mice show decreased transcript levels of GR targets, coupled with lower nuclear levels of total and phosphorylated on Ser211, GR protein. Acute DEX treatment and 24-h fasting both failed to re-activate expression of GR targets in the livers of Vps15 mutant mice to the levels observed in controls. Decreased levels of endogenous GR ligand corticosterone and lower expression of 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), a metabolic enzyme that controls corticosterone availability, were found in the livers of Vps15 mutants. Hepatic Vps15 depletion resulted in the activation of nuclear Akt1 signalling, which was paralleled by increased polyubiquitination of GR. CONCLUSION: In the liver, class 3 PI3K is required for corticosterone metabolism and GR transcriptional activity.

ATGL-dependent white adipose tissue lipolysis controls hepatocyte PPARα activity.

In hepatocytes, peroxisome proliferator-activated receptor α (PPARα) orchestrates a genomic and metabolic response required for homeostasis during fasting. This includes the biosynthesis of ketone bodies and of fibroblast growth factor 21 (FGF21). Here we show that in the absence of adipose triglyceride lipase (ATGL) in adipocytes, ketone body and FGF21 production is impaired upon fasting. Liver gene expression analysis highlights a set of fasting-induced genes sensitive to both ATGL deletion in adipocytes and PPARα deletion in hepatocytes. Adipose tissue lipolysis induced by activation of the ß3-adrenergic receptor also triggers such PPARα-dependent responses not only in the liver but also in brown adipose tissue (BAT). Intact PPARα activity in hepatocytes is required for the cross-talk between adipose tissues and the liver during fat mobilization.

mTORbeta splicing isoform promotes cell proliferation and tumorigenesis.

The mTOR (mammalian target of rapamycin) promotes growth in response to nutrients and growth factors and is deregulated in numerous pathologies, including cancer. The mechanisms by which mTOR senses and regulates energy metabolism and cell growth are relatively well understood, whereas the molecular events underlining how it mediates survival and proliferation remain to be elucidated. Here, we describe the existence of the mTOR splicing isoform, TOR beta, which, in contrast to the full-length protein (mTOR alpha), has the potential to regulate the G(1) phase of the cell cycle and to stimulate cell proliferation. mTOR beta is an active protein kinase that mediates downstream signaling through complexing with Rictor and Raptor proteins. Remarkably, overexpression of mTOR beta transforms immortal cells and is tumorigenic in nude mice and therefore could be a proto-oncogene.

CoA Synthase is phosphorylated on tyrosines in mammalian cells, interacts with and is dephosphorylated by Shp2PTP.

CoA Synthase (CoASy, 4'-phosphopantetheine adenylyltransferase/dephospho-CoA kinase) mediates two final stages of de novo coenzyme A (CoA) biosynthesis in higher eukaryotes. Unfortunately very little is known about regulation of this important metabolic pathway. In this study, we demonstrate that CoASy interacts in vitro with Src homology-2 (SH2) domains of a number of signaling proteins, including Src homology-2 domains containing protein tyrosine phosphatase (Shp2PTP). Complexes between CoASy and Shp2PTP exist in vivo in mammalian cells and this interaction is regulated in a growth-factor-dependent manner. We have also demonstrated that endogenous CoASy is phosphorylated on tyrosine residues in vivo, and that cytoplasmic protein tyrosine kinases can mediate this phosphorylation in vitro and in vivo. Importantly, Shp2PTP-mediated CoASy in vitro dephosphorylation leads to an increase in CoASy enzymatic phosphopantetheine adenylyltransferase (PPAT) activity. We therefore argue that CoASy is a novel potential substrate of Shp2PTP and phosphorylation of CoASy at tyrosine residue(s) could represent unrecognized before mechanism of modulation intracellular CoA level in response to hormonal and (or) other extracellular stimuli.

CoA synthase is in complex with p85alphaPI3K and affects PI3K signaling pathway.

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85alphaPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85alphaPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110alpha and regulatory p85alpha subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85alphaPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.

The class 3 PI3K coordinates autophagy and mitochondrial lipid catabolism by controlling nuclear receptor PPARα.

The class 3 phosphoinositide 3-kinase (PI3K) is required for lysosomal degradation by autophagy and vesicular trafficking, assuring nutrient availability. Mitochondrial lipid catabolism is another energy source. Autophagy and mitochondrial metabolism are transcriptionally controlled by nutrient sensing nuclear receptors. However, the class 3 PI3K contribution to this regulation is unknown. We show that liver-specific inactivation of Vps15, the essential regulatory subunit of the class 3 PI3K, elicits mitochondrial depletion and failure to oxidize fatty acids. Mechanistically, transcriptional activity of Peroxisome Proliferator Activated Receptor alpha (PPARα), a nuclear receptor orchestrating lipid catabolism, is blunted in Vps15-deficient livers. We find PPARα repressors Histone Deacetylase 3 (Hdac3) and Nuclear receptor co-repressor 1 (NCoR1) accumulated in Vps15-deficient livers due to defective autophagy. Activation of PPARα or inhibition of Hdac3 restored mitochondrial biogenesis and lipid oxidation in Vps15-deficient hepatocytes. These findings reveal roles for the class 3 PI3K and autophagy in transcriptional coordination of mitochondrial metabolism.

Alteration of splicing factors' expression during liver disease progression: impact on hepatocellular carcinoma outcome.

PURPOSE: Trans-acting splicing factors (SF) shape the eukaryotic transcriptome by regulating alternative splicing (AS). This process is recurrently modulated in liver cancer suggesting its direct contribution to the course of liver disease. The aim of our study was to investigate the relationship between the regulation of SFs expression and liver damage. METHODS: The expression profile of 10 liver-specific SF and the AS events of 7 genes associated with liver disorders was assessed by western-blotting in 6 murine models representing different stages of liver damage, from inflammation to hepatocellular carcinoma (HCC). Relevant SFs (PSF, SRSF3, and SRSF6) and target genes (INSR, SRSF3, and SLK) modulated in mice were investigated in a cohort of 179 HCC patients. RESULTS: Each murine model of liver disease was characterized by a unique SF expression profile. Changes in the SF profile did not affect AS events of the selected genes despite the presence of corresponding splicing sites. In human HCC expression of SFs, including the tumor-suppressor SRSF3, and AS regulation of genes studied were frequently upregulated in tumor versus non-tumor tissues. Risk of tumor recurrence positively correlated with AS isoform of the INSR gene. In contrast, increased levels of SFs expression correlated with an extended overall survival of patients. CONCLUSIONS: Dysregulation of SF expression is an early event occurring during liver injury and not just at the stage of HCC. Besides impacting on AS regulation, overexpression of SF may contribute to preserving hepatocyte homeostasis during liver pathogenesis.

A2 isoform of mammalian translation factor eEF1A displays increased tyrosine phosphorylation and ability to interact with different signalling molecules.

The eEF1A1 and eEF1A2 isoforms of translation elongation factor 1A have 98% similarity and perform the same protein synthesis function catalyzing codon-dependent binding of aminoacyl-tRNA to 80S ribosome. However, the isoforms apparently play different non-canonical roles in apoptosis and cancer development which are awaiting further investigations. We hypothesize that the difference in non-translational functions could be caused, in particular, by differential ability of the isoforms to be involved in phosphotyrosine-mediated signalling. The ability of eEF1A1 and eEF1A2 to interact with SH2 and SH3 domains of different signalling molecules in vitro was compared. Indeed, contrary to eEF1A1, eEF1A2 was able to interact with SH2 domains of Grb2, RasGAP, Shc and C-terminal part of Shp2 as well as with SH3 domains of Crk, Fgr, Fyn and phospholipase C-gamma1. Interestingly, the interaction of both isoforms with Shp2 in vivo was found using stable cell lines expressing eEF1A1-His or eEF1A2-His. The formation of a complex between endogenous eEF1A and Shp2 was also shown. Importantly, a higher level of tyrosine phosphorylation of eEF1A2 as compared to eEF1A1 was demonstrated in several independent experiments and its importance for interaction of eEF1A2 with Shp2 in vitro was revealed. Thus, despite the fact that both isoforms of eEF1A could be involved in the phosphotyrosine-mediated processes, eEF1A2 apparently has greater potential to participate in such signalling pathways. Since tyrosine kinases/phosphatases play a prominent role in human cancerogenesis, our observations may gave a basis for recently found oncogenicity of the eEF1A2 isoform.

Ribosomal protein S6 kinase 1 interacts with and is ubiquitinated by ubiquitin ligase ROC1.

Ribosomal protein S6 kinase (S6K) is involved in the regulation of cell growth and cellular metabolism. The activation of S6K in response to diverse extracellular stimuli is mediated by multiple phosphorylations coordinated by the mTOR and PI3K signaling pathways. We have recently found that both forms of S6K are modified by ubiquitination. Following these findings, we demonstrate here for the first time that S6K1 associates specifically with ubiquitin ligase ROC1 in vitro and in vivo. The interaction was initially identified in the yeast two-hybrid screening and further confirmed by pull-down and co-immunoprecipitation assays. Furthermore, the overexpression of ROC1 leads to an increase in S6K1 ubiquitination. Consistent with this observation, we showed that the steady-state level of S6K1 is regulated by ROC1, since downregulation of ROC1 by specific siRNA promotes stabilization of S6K1 protein. The results suggest the involvement of ROC1 in S6K1 ubiquitination and subsequent proteasomal degradation.

Regulation of ribosomal protein S6 kinases by ubiquitination.

Ribosomal protein S6 kinase (S6K) is a key player in the regulation of cell growth and energy metabolism via the mTOR and PI3K signalling pathways. The activity and subcellular localization of S6K are regulated by multiple S/T phosphorylations in response to diverse extracellular stimuli. Downregulation of S6K signalling occurs through the action of S/T phosphatases (PP2A and PP1) and tumor suppressors (TSC1/2 and PTEN). We report here that, in addition to phosphorylation, S6Ks are ubiquitinated in cells. The pattern of ubiquitination and the effect of proteasomal inhibitors on the steady-state level of transiently overexpressed and endogenous S6Ks point to proteasome-mediated degradation of ubiquitinated S6Ks. Furthermore, we found that the site(s) of ubiquitination are located in the kinase domain and that the N- and C-terminal regulatory regions modulate the efficiency of S6K ubiquitination. This study suggests that S6K signalling also could be regulated through the proteasome-mediated turnover of S6Ks.

Hepatocyte nuclear factor 1α suppresses steatosis-associated liver cancer by inhibiting PPARγ transcription.

Worldwide epidemics of metabolic diseases, including liver steatosis, are associated with an increased frequency of malignancies, showing the highest positive correlation for liver cancer. The heterogeneity of liver cancer represents a clinical challenge. In liver, the transcription factor PPARγ promotes metabolic adaptations of lipogenesis and aerobic glycolysis under the control of Akt2 activity, but the role of PPARγ in liver tumorigenesis is unknown. Here we have combined preclinical mouse models of liver cancer and genetic studies of a human liver biopsy atlas with the aim of identifying putative therapeutic targets in the context of liver steatosis and cancer. We have revealed a protumoral interaction of Akt2 signaling with hepatocyte nuclear factor 1α (HNF1α) and PPARγ, transcription factors that are master regulators of hepatocyte and adipocyte differentiation, respectively. Akt2 phosphorylates and inhibits HNF1α, thus relieving the suppression of hepatic PPARγ expression and promoting tumorigenesis. Finally, we observed that pharmacological inhibition of PPARγ is therapeutically effective in a preclinical murine model of steatosis-associated liver cancer. Taken together, our studies in humans and mice reveal that Akt2 controls hepatic tumorigenesis through crosstalk between HNF1α and PPARγ.

Mutations in the X-linked ATP6AP2 cause a glycosylation disorder with autophagic defects.

The biogenesis of the multi-subunit vacuolar-type H+-ATPase (V-ATPase) is initiated in the endoplasmic reticulum with the assembly of the proton pore V0, which is controlled by a group of assembly factors. Here, we identify two hemizygous missense mutations in the extracellular domain of the accessory V-ATPase subunit ATP6AP2 (also known as the [pro]renin receptor) responsible for a glycosylation disorder with liver disease, immunodeficiency, cutis laxa, and psychomotor impairment. We show that ATP6AP2 deficiency in the mouse liver caused hypoglycosylation of serum proteins and autophagy defects. The introduction of one of the missense mutations into Drosophila led to reduced survival and altered lipid metabolism. We further demonstrate that in the liver-like fat body, the autophagic dysregulation was associated with defects in lysosomal acidification and mammalian target of rapamycin (mTOR) signaling. Finally, both ATP6AP2 mutations impaired protein stability and the interaction with ATP6AP1, a member of the V0 assembly complex. Collectively, our data suggest that the missense mutations in ATP6AP2 lead to impaired V-ATPase assembly and subsequent defects in glycosylation and autophagy.

Receptor association and tyrosine phosphorylation of S6 kinases.

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.