Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.
Hsin-Wei Liao, Jung-Mao Hsu, Weiya Xia, Hung-Ling Wang, Ying-Nai Wang, Wei-Chao Chang, Stefan T. Arold, Chao-Kai Chou, Pei-Hsiang Tsou, Hirohito Yamaguchi, Yueh-Fu Fang, Hong-Jen Lee, Heng-Huan Lee, Shyh-Kuan Tai, Mhu-Hwa Yang, Maria P. Morelli, Malabika Sen, John E. Ladbury, Chung-Hsuan Chen, Jennifer R. Grandis, Scott Kopetz, Mien-Chie Hung
Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from
Yangfan P. Liu, I-Chun Tsai, Manuela Morleo, Edwin C. Oh, Carmen C. Leitch, Filomena Massa, Byung-Hoon Lee, David S. Parker, Daniel Finley, Norann A. Zaghloul, Brunella Franco, Nicholas Katsanis
Cleft lip, which results from impaired facial process growth and fusion, is one of the most common craniofacial birth defects. Many genes are known to be involved in the etiology of this disorder; however, our understanding of cleft lip pathogenesis remains incomplete. In the present study, we uncovered a role for sonic hedgehog (SHH) signaling during lip fusion. Mice carrying compound mutations in hedgehog acyltransferase (
Hiroshi Kurosaka, Angelo Iulianella, Trevor Williams, Paul A. Trainor
Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated
Kazusaku Kamiya, Sabrina W. Yum, Nagomi Kurebayashi, Miho Muraki, Kana Ogawa, Keiko Karasawa, Asuka Miwa, Xueshui Guo, Satoru Gotoh, Yoshinobu Sugitani, Hitomi Yamanaka, Shioko Ito-Kawashima, Takashi Iizuka, Takashi Sakurai, Tetsuo Noda, Osamu Minowa, Katsuhisa Ikeda
Mutations in the gene centrosomal protein 290 kDa (
Theodore G. Drivas, Erika L.F. Holzbaur, Jean Bennett
Dysfunctional telomeres limit cellular proliferative capacity by activating the p53-p21– and p16INK4a-Rb–dependent DNA damage responses (DDRs). The p16INK4a tumor suppressor accumulates in aging tissues, is a biomarker for cellular senescence, and limits stem cell function in vivo. While the activation of a p53-dependent DDR by dysfunctional telomeres has been well documented in human cells and mouse models, the role for p16INK4a in response to telomere dysfunction remains unclear. Here, we generated protection of telomeres 1b
Yang Wang, Norman Sharpless, Sandy Chang
Aberrant regulation of eNOS and associated NO release are directly linked with various vascular diseases. Caveolin-1 (Cav-1), the main coat protein of caveolae, is highly expressed in endothelial cells. Its scaffolding domain serves as an endogenous negative regulator of eNOS function. Structure-function analysis of Cav-1 has shown that phenylalanine 92 (F92) is critical for the inhibitory actions of Cav-1 toward eNOS. Herein, we show that F92A–Cav-1 and a mutant cell–permeable scaffolding domain peptide called Cavnoxin can increase basal NO release in eNOS-expressing cells. Cavnoxin reduced vascular tone ex vivo and lowered blood pressure in normal mice. In contrast, similar experiments performed with eNOS- or Cav-1–deficient mice showed that the vasodilatory effect of Cavnoxin is abolished in the absence of these gene products, which indicates a high level of eNOS/Cav-1 specificity. Mechanistically, biochemical assays indicated that noninhibitory F92A–Cav-1 and Cavnoxin specifically disrupted the inhibitory actions of endogenous Cav-1 toward eNOS and thereby enhanced basal NO release. Collectively, these data raise the possibility of studying the inhibitory influence of Cav-1 on eNOS without interfering with the other actions of endogenous Cav-1. They also suggest a therapeutic application for regulating the eNOS/Cav-1 interaction in diseases characterized by decreased NO release.
Pascal Bernatchez, Arpeeta Sharma, Philip M. Bauer, Ethan Marin, William C. Sessa
GPCR inhibitors are highly prevalent in modern therapeutics. However, interference with complex GPCR regulatory mechanisms leads to both therapeutic efficacy and adverse effects. Recently, the sphingosine-1-phosphate (S1P) receptor inhibitor FTY720 (also known as Fingolimod), which induces lymphopenia and prevents neuroinflammation, was adopted as a disease-modifying therapeutic in multiple sclerosis. Although highly efficacious, dose-dependent increases in adverse events have tempered its utility. We show here that FTY720P induces phosphorylation of the C-terminal domain of S1P receptor 1 (S1P1) at multiple sites, resulting in GPCR internalization, polyubiquitinylation, and degradation. We also identified the ubiquitin E3 ligase WWP2 in the GPCR complex and demonstrated its requirement in FTY720-induced receptor degradation. GPCR degradation was not essential for the induction of lymphopenia, but was critical for pulmonary vascular leak in vivo. Prevention of receptor phosphorylation, internalization, and degradation inhibited vascular leak, which suggests that discrete mechanisms of S1P receptor regulation are responsible for the efficacy and adverse events associated with this class of therapeutics.
Myat Lin Oo, Sung-Hee Chang, Shobha Thangada, Ming-Tao Wu, Karim Rezaul, Victoria Blaho, Sun-Il Hwang, David K. Han, Timothy Hla
Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro-translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft–associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endosomal sorting of the toxin-GM1 complex.
David E. Saslowsky, Jin Ah Cho, Himani Chinnapen, Ramiro H. Massol, Daniel J.-F. Chinnapen, Jessica S. Wagner, Heidi E. De Luca, Wendy Kam, Barry H. Paw, Wayne I. Lencer
Hereditary spastic paraplegias (HSPs; SPG1–45) are inherited neurological disorders characterized by lower extremity spastic weakness. More than half of HSP cases result from autosomal dominant mutations in atlastin-1 (also known as SPG3A), receptor expression enhancing protein 1 (REEP1; SPG31), or spastin (SPG4). The atlastin-1 GTPase interacts with spastin, a microtubule-severing ATPase, as well as with the DP1/Yop1p and reticulon families of ER-shaping proteins, and SPG3A caused by atlastin-1 mutations has been linked pathogenically to abnormal ER morphology. Here we investigated SPG31 by analyzing the distribution, interactions, and functions of REEP1. We determined that REEP1 is structurally related to the DP1/Yop1p family of ER-shaping proteins and localizes to the ER in cultured rat cerebral cortical neurons, where it colocalizes with spastin and atlastin-1. Upon overexpression in COS7 cells, REEP1 formed protein complexes with atlastin-1 and spastin within the tubular ER, and these interactions required hydrophobic hairpin domains in each of these proteins. REEP proteins were required for ER network formation in vitro, and REEP1 also bound microtubules and promoted ER alignment along the microtubule cytoskeleton in COS7 cells. A SPG31 mutant REEP1 lacking the C-terminal cytoplasmic region did not interact with microtubules and disrupted the ER network. These data indicate that the HSP proteins atlastin-1, spastin, and REEP1 interact within the tubular ER membrane in corticospinal neurons to coordinate ER shaping and microtubule dynamics. Thus, defects in tubular ER shaping and network interactions with the microtubule cytoskeleton seem to be the predominant pathogenic mechanism of HSP.
Seong H. Park, Peng-Peng Zhu, Rell L. Parker, Craig Blackstone
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