Inhibition of Pol I transcription treats murine and human AML by targeting the leukemia-initiating cell population.

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs.

Inositol polyphosphate 4-phosphatase II (INPP4B) is associated with chemoresistance and poor outcome in AML.

Phosphoinositide signaling regulates diverse cellular functions. Phosphoinositide-3 kinase (PI3K) generates PtdIns(3,4,5)P3 and PtdIns(3,4)P2, leading to the activation of proliferative and anti-apoptotic signaling pathways. Termination of phosphoinositide signaling requires hydrolysis of inositol ring phosphate groups through the actions of PtdIns(3,4,5)P3 3-phosphatase (PTEN), PtdIns(3,4,5)P3 5-phosphatases (eg, SHIP), and PtdIns(3,4)P2 4-phosphatases (eg, INPP4B). The biological relevance of most of these phosphoinositide phosphatases in acute myeloid leukemia (AML) remains poorly understood. Mass spectrometry-based gene expression profiling of 3-, 4- and 5-phosphatases in human AML revealed significant overexpression of INPP4B. Analysis of an expanded panel of 205 AML cases at diagnosis revealed INPP4B overexpression in association with reduced responses to chemotherapy, early relapse, and poor overall survival, independent of other risk factors. Ectopic overexpression of INPP4B conferred leukemic resistance to cytosine arabinoside (ara-C), daunorubicin, and etoposide. Expression of a phosphatase inert variant (INPP4B C842A) failed to abrogate resistance of AML cells to chemotherapy in vitro or in vivo. In contrast, targeted suppression of endogenously overexpressed INPP4B by RNA interference sensitized AML cell lines and primary AML to chemotherapy. These findings demonstrate a previously unsuspected and clinically relevant role for INPP4B gain of function as a mediator of chemoresistance and poor survival outcome in AML independent of its phosphoinositide phosphatase function.

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.

Targeting acute myeloid leukemia by dual inhibition of PI3K signaling and Cdk9-mediated Mcl-1 transcription.

Resistance to cell death is a hallmark of cancer and renders transformed cells resistant to multiple apoptotic triggers. The Bcl-2 family member, Mcl-1, is a key driver of cell survival in diverse cancers, including acute myeloid leukemia (AML). A screen for compounds that downregulate Mcl-1 identified the kinase inhibitor, PIK-75, which demonstrates marked proapoptotic activity against a panel of cytogenetically diverse primary human AML patient samples. We show that PIK-75 transiently blocks Cdk7/9, leading to transcriptional suppression of MCL-1, rapid loss of Mcl-1 protein, and alleviation of its inhibition of proapoptotic Bak. PIK-75 also targets the p110α isoform of PI3K, which leads to a loss of association between Bcl-xL and Bak. The simultaneous loss of Mcl-1 and Bcl-xL association with Bak leads to rapid apoptosis of AML cells. Concordantly, low Bak expression in AML confers resistance to PIK-75-mediated killing. On the other hand, the induction of apoptosis by PIK-75 did not require the expression of the BH3 proteins Bim, Bid, Bad, Noxa, or Puma. PIK-75 significantly reduced leukemia burden and increased the survival of mice engrafted with human AML without inducing overt toxicity. Future efforts to cotarget PI3K and Cdk9 with drugs such as PIK-75 in AML are warranted.

Discovery and SAR of novel pyrazolo[1,5-a]pyrimidines as inhibitors of CDK9.

The serine-threonine kinase CDK9 is a target of emerging interest for the development of anti-cancer drugs. There are multiple lines of evidence linking CDK9 activity to cancer, including the essential role this kinase plays in transcriptional regulation through phosphorylation of the C-terminal domain (CTD) of RNA polymerase II. Indeed, inhibition of CDK9 has been shown to result in a reduction of short-lived proteins such as the pro-survival protein Mcl-1 in malignant cells leading to the induction of apoptosis. In this work we report our initial studies towards the discovery of selective CDK9 inhibitors, starting from the known multi-kinase inhibitor PIK-75 which possesses potent CDK9 activity. Our series is based on a pyrazolo[1,5-a]pyrimidine nucleus and, importantly, the resultant lead compound 18b is devoid of the structural liabilities present in PIK-75 and possesses greater selectivity.

Phosphorylation of serine 779 in fibroblast growth factor receptor 1 and 2 by protein kinase C(epsilon) regulates Ras/mitogen-activated protein kinase signaling and neuronal differentiation.

The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser(779) in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser(779) in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser(779) was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCε can phosphorylate Ser(779) in vitro, whereas overexpression of PKCε results in constitutive Ser(779) phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCε reduces both growth factor-induced Ser(779) phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser(779), can quantitatively control Ras/MAPK signaling to promote specific cellular responses.

New insights into the nutritional genomics of adult-onset riboflavin-responsive diseases.

Riboflavin, or vitamin B2, is an essential nutrient that serves as a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). The binding of the FAD and/or FMN cofactors to flavoproteins is critical for regulating their assembly and activity. There are over 90 proteins in the human flavoproteome that regulate a diverse array of biochemical pathways including mitochondrial metabolism, riboflavin transport, ubiquinone and FAD synthesis, antioxidant signalling, one-carbon metabolism, nitric oxide signalling and peroxisome oxidative metabolism. The identification of patients with genetic variants in flavoprotein genes that lead to adult-onset pathologies remains a major diagnostic challenge. However, once identified, many patients with adult-onset inborn errors of metabolism demonstrate remarkable responses to riboflavin therapy. We review the structure:function relationships of mutant flavoproteins and propose new mechanistic insights into adult-onset riboflavin-responsive pathologies and metabolic dysregulations that apply to multiple biochemical pathways. We further address the vexing issue of how the inheritance of genetic variants in flavoprotein genes leads to an adult-onset disease with complex symptomologies and varying severities. We also propose a broad clinical framework that may not only improve the current diagnostic rates, but also facilitate a personalized approach to riboflavin therapy that is low cost, safe and lead to transformative outcomes in many patients.

The granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signaling and its role in disease.

Already 20 years have passed since the cloning of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, the first member of the GM-CSF/interleukin (IL)-3/IL-5 family of hemopoietic cytokine receptors to be molecularly characterized. The intervening 2 decades have uncovered a plethora of biologic functions transduced by the GM-CSF receptor (pleiotropy) and revealed distinct signaling networks that couple the receptor to biologic outcomes. Unlike other hemopoietin receptors, the GM-CSF receptor has a significant nonredundant role in myeloid hematologic malignancies, macrophage-mediated acute and chronic inflammation, pulmonary homeostasis, and allergic disease. The molecular mechanisms underlying GM-CSF receptor activation have recently been revealed by the crystal structure of the GM-CSF receptor complexed to GM-CSF, which shows an unexpected higher order assembly. Emerging evidence also suggests the existence of intracellular signosomes that are recruited in a concentration-dependent fashion to selectively control cell survival, proliferation, and differentiation by GM-CSF. These findings begin to unravel the mystery of cytokine receptor pleiotropy and are likely to also apply to the related IL-3 and IL-5 receptors as well as other heterodimeric cytokine receptors. The new insights in GM-CSF receptor activation have clinical significance as the structural and signaling nuances can be harnessed for the development of new treatments for malignant and inflammatory diseases.

Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML.

Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the betac subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene betac Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34(+)CD38(-)CD123(+) leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI-TOF/TOF MS.

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high-sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti-phosphotyrosine antibody 4G10 was coupled covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non-phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro-phosphorylated recombinant beta-intracellular region of the granulocyte-macrophage colony-stimulating factor receptor, which was subsequently analysed by MALDI-TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.

Meeting report: Barossa 2005--Signaling Networks.

Cellular signal transduction involves an elaborate network of interrelated signaling pathways. Dissecting the components of these signaling pathways and the functional relationships between them is crucial to our understanding of biological processes. This was the central theme of the November 2005 Signaling Networks meeting held in the Barossa Valley, South Australia. The meeting highlighted recent exciting advances in this area, covering topics such as the initiation, integration, regulation, and architecture of signaling networks, and the importance of these pathways in normal physiological functions and pathophysiological processes.

Activation of protein phosphatase 2A in FLT3+ acute myeloid leukemia cells enhances the cytotoxicity of FLT3 tyrosine kinase inhibitors.

Constitutive activation of the receptor tyrosine kinase Fms-like tyrosine kinase 3 (FLT3), via co-expression of its ligand or by genetic mutation, is common in acute myeloid leukemia (AML). In this study we show that FLT3 activation inhibits the activity of the tumor suppressor, protein phosphatase 2A (PP2A). Using BaF3 cells transduced with wildtype or mutant FLT3, we show that FLT3-induced PP2A inhibition sensitizes cells to the pharmacological PP2A activators, FTY720 and AAL(S). FTY720 and AAL(S) induced cell death and inhibited colony formation of FLT3 activated cells. Furthermore, PP2A activators reduced the phosphorylation of ERK and AKT, downstream targets shared by both FLT3 and PP2A, in FLT3/ITD+ BaF3 and MV4-11 cell lines. PP2A activity was lower in primary human bone marrow derived AML blasts compared to normal bone marrow, with blasts from FLT3-ITD patients displaying lower PP2A activity than WT-FLT3 blasts. Reduced PP2A activity was associated with hyperphosphorylation of the PP2A catalytic subunit, and reduced expression of PP2A structural and regulatory subunits. AML patient blasts were also sensitive to cell death induced by FTY720 and AAL(S), but these compounds had minimal effect on normal CD34+ bone marrow derived monocytes. Finally, PP2A activating compounds displayed synergistic effects when used in combination with tyrosine kinase inhibitors in FLT3-ITD+ cells. A combination of Sorafenib and FTY720 was also synergistic in the presence of a protective stromal microenvironment. Thus combining a PP2A activating compound and a FLT3 inhibitor may be a novel therapeutic approach for treating AML.

14-3-3ζ coordinates adipogenesis of visceral fat.

The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27(Kip1). Indeed, concomitant knockdown of p27(Kip1) or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27(Kip1) abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis.

Oncogenic mutations of p110α isoform of PI 3-kinase upregulate its protein kinase activity.

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 ßc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 ßc receptor (ßic). We find p110α, p110ß and p110γ all phosphorylate ßic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards ßic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn(2+) dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110ß and p110γ all display significant activity in the presence of Mg(2+). Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.

Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers.

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55alpha, B56alpha, B56gamma, and B56delta). Overexpression of PP2A-Aalpha in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT(+) cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT(+) cancers.

14-3-3:Shc scaffolds integrate phosphoserine and phosphotyrosine signaling to regulate phosphatidylinositol 3-kinase activation and cell survival.

Integrated cascades of protein tyrosine and serine/threonine phosphorylation play essential roles in transducing signals in response to growth factors and cytokines. How adaptor or scaffold proteins assemble signaling complexes through both phosphotyrosine and phosphoserine/threonine residues to regulate specific signaling pathways and biological responses is unclear. We show in multiple cell types that endogenous 14-3-3zeta is phosphorylated on Tyr(179) in response to granulocyte macrophage colony-stimulating factor. Importantly, 14-3-3zeta can function as an intermolecular bridge that couples to phosphoserine residues and also directly binds the SH2 domain of Shc via Tyr(179). The assembly of these 14-3-3:Shc scaffolds is specifically required for the recruitment of a phosphatidylinositol 3-kinase signaling complex and the regulation of CTL-EN cell survival in response to cytokine. The biological significance of these findings was further demonstrated using primary bone marrow-derived mast cells from 14-3-3zeta(-/-) mice. We show that cytokine was able to promote Akt phosphorylation and viability of primary mast cells derived from 14-3-3zeta(-/-) mice when reconstituted with wild type 14-3-3zeta, but the Akt phosphorylation and survival response was reduced in cells reconstituted with the Y179F mutant. Together, these results show that 14-3-3:Shc scaffolds can act as multivalent signaling nodes for the integration of both phosphoserine/threonine and phosphotyrosine pathways to regulate specific cellular responses.

Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells.

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Fibroblast growth factor receptor 2 phosphorylation on serine 779 couples to 14-3-3 and regulates cell survival and proliferation.

The fibroblast growth factors (FGFs) exert their diverse (or pleiotropic) biological responses through the binding and activation of specific cell surface receptors (FGFRs). While FGFRs are known to initiate intracellular signaling through receptor tyrosine phosphorylation, the precise mechanisms by which the FGFRs regulate pleiotropic biological responses remain unclear. We now identify a new mechanism by which FGFR2 is able to regulate intracellular signaling and cellular responses. We show that FGFR2 is phosphorylated on serine 779 (S779) in response to FGF2. S779, which lies adjacent to the phospholipase Cgamma binding site at Y766, provides a docking site for the 14-3-3 phosphoserine-binding proteins and is essential for the full activation of the phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase pathways. Furthermore, S779 signaling is essential for promoting cell survival and proliferation in both Ba/F3 cells and BALB/c 3T3 fibroblasts. This new mode of FGFR2 phosphoserine signaling via the 14-3-3 proteins may provide an increased repertoire of signaling outputs to allow the regulation of pleiotropic biological responses. In this regard, we have identified conserved putative phosphotyrosine/phosphoserine motifs in the cytoplasmic domains of diverse cell surface receptors, suggesting that they may perform important functional roles beyond the FGFRs.

The Shc-binding site of the betac subunit of the GM-CSF/IL-3/IL-5 receptors is a negative regulator of hematopoiesis.

Tyrosine and serine phosphorylation of the common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is widely viewed as a general mechanism that provides positive inputs by coupling the receptor to signaling pathways that stimulate several cellular functions. We show here that despite the known action of Tyr577 in beta(c) to recruit Shc-PI-3 kinase (PI3K) pathway members, Tyr577 plays, surprisingly, a negative regulatory role in cell function, and that this is mediated, at least in part, through the uncoupling of SH2-containing inositol 5'-phosphatase (SHIP) from beta(c). Fetal liver cells from beta(c)/beta(IL-3)(-/-) mice expressing human GM-CSF receptor alpha chain and beta(c) Tyr577Phe mutant showed enhanced colony formation and expansion of progenitor cells in response to GM-CSF. Dissection of these activities revealed that basal survival was increased, as well as cytokine-stimulated proliferation. As expected, the recruitment and activation of Shc was abolished, but interestingly, Gab-2 and Akt phosphorylation increased. Significantly, the activation of PI3K was enhanced and prolonged, accompanied by loss of SHIP activity. These results reveal a previously unrecognized negative signaling role for Tyr577 in beta(c) and demonstrate that uncoupling Shc from cytokine receptors enhances PI3K signaling as well as survival and proliferation.

Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch.

Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage-colony-stimulating factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems.