Although dominant gain-of-function triplet repeat expansions in the Huntingtin (HTT) gene are the underlying cause of Huntington disease (HD), understanding the normal functions of nonmutant HTT protein has remained a challenge. We report here findings that suggest that HTT plays a significant role in selective autophagy. Loss of HTT function in Drosophila disrupts starvationinduced autophagy in larvae and conditional knockout of HTT in the mouse CNS causes characteristic cellular hallmarks of disrupted autophagy, including an accumulation of striatal p62/SQSTM1 over time. We observe that specific domains of HTT have structural similarities to yeast Atg proteins that function in selective autophagy, and in particular that the C-terminal domain of HTT shares structural similarity to yeast Atg11, an autophagic scaffold protein. To explore possible functional similarity between HTT and Atg11, we investigated whether the C-terminal domain of HTT interacts with mammalian counterparts of yeast Atg11-interacting proteins. Strikingly, this domain of HTT coimmunoprecipitates with several key Atg11 interactors, including the Atg1/Unc-51-like autophagy activating kinase 1 kinase complex, autophagic receptor proteins, and mammalian Atg8 homologs. Mutation of a phylogenetically conserved WXXL domain in a C-terminal HTT fragment reduces coprecipitation with mammalian Atg8 homolog GABARAPL1, suggesting a direct interaction. Collectively, these data support a possible central role for HTT as an Atg11-like scaffold protein. These findings have relevance to both mechanisms of disease pathogenesis and to therapeutic intervention strategies that reduce levels of both mutant and normal HTT.rodegenerative disorder caused by an expansion of a CAG trinucleotide repeat encoding a polyglutamine (polyQ) tract near the N terminus of the 350-kD Huntingtin protein (HTT) (1). Identifying the normal biological function of the HTT protein is important in the effort to design and implement effective therapeutic interventions for HD, but has proved challenging.In the mouse, loss of HTT leads to lethality during gastrulation at embryonic day 7 (2-4). Conditional inactivation of HTT in the mouse forebrain at postnatal or late embryonic stages causes a progressive neurodegenerative phenotype associated with neuronal degeneration, motor phenotypes, and early mortality (5). Loss of HTT in mouse cells reduces primary cilia formation, and deletion of HTT in ependymal cells leads to alteration of the cilia layer, suggesting a role for HTT in ciliogenesis (6). Mutant HTT expression and HTT knockdown have also been found to impair axonal trafficking of vesicles, mitochondria, and autophagosomes in neurons in vitro and in vivo (7-9). A clear molecular mechanism to relate these findings to the function of the HTT protein, however, has not yet emerged.In contrast to the embryonic lethality observed in the mouse, Drosophila lacking the endogenous Htt gene develop normally. However, adult Drosophila HTT loss-of-function (LOF) flies show an accelerated neurodege...

Neural cultures derived from Huntington’s disease (HD) patient-derived induced pluripotent stem cells were used for ‘omics’ analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling, axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development, the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks, gene expression and unique peak profiles associated with dysregulated genes, suggesting a coordinated epigenetic program. Treatment with isoxazole-9, which targets key dysregulated pathways, led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time.

Receptor tyrosine kinase signaling cooperates with WNT/β-catenin signaling in regulating many biological processes, but the mechanisms of their interaction remain poorly defined. We describe a potent activation of WNT/β-catenin by FGFR2, FGFR3, EGFR and TRKA kinases, which is independent of the PI3K/AKT pathway. Instead, this phenotype depends on ERK MAP kinase-mediated phosphorylation of WNT co-receptor LRP6 at Ser1490 and Thr1572 during its Golgi network-based maturation process. This phosphorylation dramatically increases the cellular response to WNT. Moreover, FGFR2, FGFR3, EGFR and TRKA directly phosphorylate β-catenin at Tyr142, which is known to increase cytoplasmic β-catenin concentration via release of β-catenin from membranous cadherin complexes. We conclude that signaling via ERK/LRP6 pathway and direct β-catenin phosphorylation at Tyr142 represent two mechanisms used by various receptor tyrosine kinase systems to activate canonical WNT signaling.

Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701) phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa) or no activation (293T and RCS). This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human skeletal dysplasias as a result of attenuation of cartilage growth. It is believed that FGFR3 inhibits chondrocyte proliferation via activation of signal transducers and activators of transcription (STAT) proteins, although the exact mechanism of both STAT activation and STAT-mediated inhibition of chondrocyte growth is unclear. We show that FGFR3 interacts with STAT1 in cells and is capable of activating phosphorylation of STAT1 in a kinase assay, thus potentially serving as a STAT1 kinase in chondrocytes. However, as demonstrated by western blotting with phosphorylation-specific antibodies, imaging of STAT nuclear translocation, STAT transcription factor assays and STAT luciferase reporter assays, FGF does not activate STAT1 or STAT3 in RCS chondrocytes, which nevertheless respond to a FGF stimulus with potent growth arrest. Moreover, addition of active STAT1 and STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation or expression of constitutively active STAT mutants does not sensitize RCS chondrocytes to FGF-mediated growth arrest. Since FGF-mediated growth arrest is rescued by siRNA-mediated downregulation of the MAP kinase ERK1/2 but not STAT1 or STAT3, our data support a model whereby the ERK arm but not STAT arm of FGF signaling in chondrocytes accounts for the growth arrest phenotype.

Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human dwarfisms characterized by diminished long bone growth (1). In cartilage, FGFR3 alters chondrocyte proliferation and differentiation by up-regulation of cell cycle inhibitors and stimulation of cartilage matrix degradation (2-5). The anti-proliferative action of FGF 2 signaling in cartilage contrasts with the usual mitogenic response of cells to FGF stimulus (6), but the molecular basis of this paradox remains unclear. Recently, Erk MAP kinase was found as a candidate for FGFR3-mediated inhibition of chondrocyte proliferation and differentiation (7-10).Protein kinase C (PKC) comprises a family of serine/threonine kinases that phosphorylate the consensus motif RXX(S/T)XR (11). The PKCs are further divided into three subfamilies based on sequence similarities and modes of activation. The conventional PKCs (PKC␣, -␤I, -␤II, and -␥) are activated by phosphatidylserine, diacylglycerol, and Ca 2ϩ , the novel PKCs (PKC␦, -⑀, -, and -) require only phosphatidylserine and diacylglycerol, and the atypical PKCs (aPKC; PKC and -) respond to phosphatidylserine alone (12). The PKC phosphorylation motif is present in many proteins (13), implicating PKCs as broad specificity protein kinases. PKCs are involved in numerous signaling events including activation of the Erk MAP kinase pathway. This is evident by potent Erk activation in cells treated with phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), which activates both conventional PKCs and novel PKCs through binding of their diacylglycerol site (14). In PMA-treated cells, PKCs target the Erk module at the level of both Raf-1 and MEK, through direct activatory phosphorylation or indirectly (15-21). Apart from PMA-mediated Erk activation, PKCs appear to be crucial for long term Erk activation by growth factors, including FGFs (20,[22][23][24][25], as well as for oncogenic .FGF signaling in chondrocytes leads to long term Ras/Erk activation, which appears to account for the growth inhibitory outcome of FGF treatment (8, 9). To date, little is known about chondrocyte properties of FGF signaling permitting prolonged Erk activity, although slow down-regulation of mutated FGFR3 appears to be involved (30,31).

Cancer is a major public health problem worldwide. In the United States alone, 1 in 4 deaths is due to cancer and for 2013 a total of 1,660,290 new cancer cases and 580,350 cancer-related deaths are projected. Comprehensive profiling of multiple cancer genomes has revealed a highly complex genetic landscape in which a large number of altered genes, varying from tumor to tumor, impact core biological pathways and processes. This has implications for therapeutic targeting of signaling networks in the development of treatments for specific cancers. The NFκB transcription factor is constitutively active in a number of hematologic and solid tumors, and many signaling pathways implicated in cancer are likely connected to NFκB activation. A critical mediator of NFκB activity is TGFβ-activated kinase 1 (TAK1). Here, we identify TAK1 as a novel interacting protein and target of fibroblast growth factor receptor 3 (FGFR3) tyrosine kinase activity. We further demonstrate that activating mutations in FGFR3 associated with both multiple myeloma and bladder cancer can modulate expression of genes that regulate NFκB signaling, and promote both NFκB transcriptional activity and cell adhesion in a manner dependent on TAK1 expression in both cancer cell types. Our findings suggest TAK1 as a potential therapeutic target for FGFR3-associated cancers, and other malignancies in which TAK1 contributes to constitutive NFκB activation.

Ectopic activation of fibroblast growth factor receptor 3 (FGFR3) is associated with several cancers, including multiple myeloma (MM). FGFR3 inhibition in these cells inhibits proliferation and induces apoptosis, validating FGFR3 signaling as a therapeutic target in t(4;14) MM cases. We have identified the PI3K regulatory subunit, p85alpha, as a novel interactor of FGFR3 by yeast two-hybrid, and confirmed an interaction with both p85alpha and p85beta in mammalian cells. The interaction of FGFR3 with p85 is dependent upon receptor activation. In contrast to the Gab1-mediated association of FGFRs with p85, the FGFR3-p85 interaction we observed requires FGFR3 Y760, previously identified as a PLCgamma binding site. The interaction of p85 with FGFR3 does not require PLCgamma, suggesting the p85 interaction is direct and independent of PLCgamma binding. FGFR3 and p85 proteins also interact in MM cell lines which consistently express p85alpha and p85beta, but not p50 or p55 subunits. siRNA knockdown of p85beta in MM cells caused an increased ERK response to FGF2. These data suggest that an endogenous negative regulatory role for the p85-FGFR3 interaction on the Ras/ERK/MAPK pathway may exist in response to FGFR3 activity and identifies a novel therapeutic target for MM.