Odin (ANKS1A) is a Src family kinase target in colorectal cancer cells

Background

Src family kinases (SFK) are implicated in the development of some colorectal cancers (CRC). One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised.

Results

64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY) proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM), GIT1, vimentin and AFAP1L2 (XB130). Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells.

Conclusion

Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.     

E-Cadherin Binding Modulates EGF Receptor Activation

We found that E-cadherin and epidermal growth factor receptor (EGFR) are associated in mammary epithelial cells and that E-cadherin engagement in these cells induces transient activation of EGFR, as previously seen in keratinocytes (37). In contrast, EGFR does not associate with and is not activated by N-cadherin. Analysis of cells expressing chimeric cadherins revealed that the extracellular domain of E-cadherin is required for interaction with and activation of EGFR. This activation results in tyrosine phosphorylation of known EGFR substrates and reduction in focal adhesions. These interactions, however, are not necessary for suppression of cell motility by E-cadherin.     

Src-Like Adaptor Protein (SLAP) Binds to the Receptor Tyrosine Kinase Flt3 and Modulates Receptor Stability and Downstream Signaling

Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.     

MICAL-L1 Links EHD1 to Tubular Recycling Endosomes and Regulates Receptor Recycling

Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.     

A GIPC1-Palmitate Switch Modulates Dopamine Drd3 Receptor Trafficking and Signaling

Palmitoylation is involved in several neuropsychiatric and movement disorders for which a dysfunctional signaling of the dopamine D3 receptor (Drd3) is hypothesized. Computational modeling of Drd3''s homologue, Drd2, has shed some light on the putative role of palmitoylation as a reversible switch for dopaminergic receptor signaling. Drd3 is presumed to be palmitoylated, based on sequence homology with Drd2, but the functional attributes afforded by Drd3 palmitoylation have not been studied. Since these receptors are major targets of antipsychotic and anti-Parkinsonian drugs, a better characterization of Drd3 signaling and posttranslational modifications, like palmitoylation, may improve the prospects for drug development. Using molecular dynamics simulations, we evaluated in silico how Drd3 palmitoylation could elicit significant remodeling of the C-terminal cytoplasmic domain to expose docking sites for signaling proteins. We tested this model in cellulo by using the interaction of Drd3 with the G-alpha interacting protein (GAIP) C terminus 1 (GIPC1) as a template. From a series of biochemical studies, live imaging, and analyses of mutant proteins, we propose that Drd3 palmitoylation acts as a molecular switch for Drd3-biased signaling via a GIPC1-dependent route, which is likely to affect the mode of action of antipsychotic drugs.     

The Cannabinoid 1 Receptor (CNR1) 1359 G/A Polymorphism Modulates Susceptibility to Ulcerative Colitis and the Phenotype in Crohn's Disease

Background

Recent evidence suggests a crucial role of the endocannabinoid system, including the cannabinoid 1 receptor (CNR1), in intestinal inflammation. We therefore investigated the influence of the CNR1 1359 G/A (p.Thr453Thr; rs1049353) single nucleotide polymorphism (SNP) on disease susceptibility and phenotype in patients with ulcerative colitis (UC) and Crohn''s disease (CD).

Methods

Genomic DNA from 579 phenotypically well-characterized individuals was analyzed for the CNR1 1359 G/A SNP. Amongst these were 166 patients with UC, 216 patients with CD, and 197 healthy controls.

Results

Compared to healthy controls, subjects A/A homozygous for the CNR1 1359 G/A SNP had a reduced risk to develop UC (p = 0.01, OR 0.30, 95% CI 0.12–0.78). The polymorphism did not modulate CD susceptibility, but carriers of the minor A allele had a lower body mass index than G/G wildtype carriers (p = 0.0005). In addition, homozygous carriers of the G allele were more likely to develop CD before 40 years of age (p = 5.9×10⁻⁷) than carriers of the A allele.

Conclusion

The CNR1 p.Thr453Thr polymorphism appears to modulate UC susceptibility and the CD phenotype. The endocannabinoid system may influence the manifestation of inflammatory bowel diseases, suggesting endocannabinoids as potential target for future therapies.     

Sigma-1 Receptor Modulates Neuroinflammation After Traumatic Brain Injury

Traumatic brain injury (TBI) remains a significant clinical problem and contributes to one-third of all injury-related deaths. Activated microglia-mediated inflammatory response is a distinct characteristic underlying pathophysiology of TBI. Here, we evaluated the effect and possible mechanisms of the selective Sigma-1 receptor agonist 2-(4-morpholinethyl)-1-phenylcyclohexanecarboxylate (PRE-084) in mice TBI model. A single intraperitoneal injection 10 μg/g PRE-084, given 15 min after TBI significantly reduced lesion volume, lessened brain edema, attenuated modified neurological severity score, increased the latency time in wire hang test, and accelerated body weight recovery. Moreover, immunohistochemical analysis with Iba1 staining showed that PRE-084 lessened microglia activation. Meanwhile, PRE-084 reduced nitrosative and oxidative stress to proteins. Thus, Sigma-1 receptors play a major role in inflammatory response after TBI and may serve as useful target for TBI treatment in the future.     

Internalization and Recycling of the CB1 Cannabinoid Receptor

Tolerance develops rapidly to cannabis, cannabinoids, and related drugs acting at the CB1 cannabinoid receptor. However, little is known about what happens to the receptor as tolerance is developing. In this study, we have found that CB1 receptors are rapidly internalized following agonist binding and receptor activation. Efficacious cannabinoid agonists (WIN 55,212-2, CP 55,940, and HU 210) caused rapid internalization. Methanandamide (an analogue of an endogenous cannabinoid, anandamide) was less effective, causing internalization only at high concentration, whereas delta9-tetrahydrocannabinol caused little internalization, even at 3 microM. CB1 internalized via clathrin-coated pits as sequestration was inhibited by hypertonic sucrose. Internalization did not require activated G protein alpha(i), alpha(o), or alpha(s) subunits. A region of the extreme carboxy terminus of the receptor was necessary for internalization, as a mutant CB1 receptor lacking the last 14 residues did not internalize, whereas a mutant lacking the last 10 residues did. Steps involved in the recycling of sequestered receptor were also investigated. Recovery of CB1 to the cell surface after short (20 min) but not long (90 min) agonist treatment was independent of new protein synthesis. Recycling also required endosomal acidification and dephosphorylation. These results show that CB1 receptor trafficking is dynamically regulated by cannabimimetic drugs.     

HECT E3 Ubiquitin Ligase Nedd4-1 Ubiquitinates ACK and Regulates Epidermal Growth Factor (EGF)-Induced Degradation of EGF Receptor and ACK

ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.Activated Cdc42-associated tyrosine kinase (ACK) (also Tnk2) is a member of the type VIII tyrosine kinase family. Activation of ACK, including both ACK1 and ACK2, occurs in response to signaling of epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF) receptor, insulin receptor, Gas-6 receptor (Mer), M3 muscarinic receptor, integrins, or proteoglycan (3, 7, 11, 23, 26, 30, 44, 47). In Drosophila, D-ACK mediates the function of Cdc42 in dorsal closure during embryonic development (31). The ACK homologue, Ark-1, in Caenorhabditis elegans negatively regulates EGF signaling (15).A number of studies suggest a role for ACK in EGFR degradation. ACK1 and ACK2, two alternatively spliced isoforms, possess a highly conserved clathrin-binding motif and interact with clathrin (37, 45). Overexpression of ACK2 severely impairs transferrin receptor endocytosis, causes aberrant localization of AP-2, and induces changes in clathrin assembly. Furthermore, ACK2 interacts with sorting nexin 9 (SNX9, also named SH3PX1), a member of the sorting nexin family, via its proline-rich domain 1 and phosphorylates SNX9 to facilitate the degradation of EGF receptors (22). In C. elegans, Ark-1 genetically interacts with UNC101, the homologue of mammalian clathrin-associated protein AP47, and SLI-1, the homologue of mammalian Cbl that is an E3 ubiquitin ligase for ubiquitination of EGFR, and negatively regulates EGFR signaling (15).Our previous studies showed that ACK1 interacts with EGFR upon EGF stimulation via a region at the carboxyl terminus, designated the EGFR-binding domain (EBD), which is highly homologous to the EGFR/ErbB2-binding domain of Gene-33/Mig-6/RALT (32, 43). The interaction of ACK1 with EGFR is dependent on kinase activity and tyrosine phosphorylation of EGFR. Immunofluorescent staining using anti-EGFR and GFP-ACK1 indicates that ACK1 is colocalized with EGFR on large vacuolar structures upon EGF stimulation. Suppression of the expression of ACK1 by ACK-RNA interference (RNAi) inhibits ligand-induced degradation of EGFR, suggesting that ACK1 plays an important role in the regulation of EGFR degradation in cells. Furthermore, we identified ACK1 as an ubiquitin-binding protein. Through an ubiquitin association (Uba) domain at the carboxyl terminus, ACK1 is capable of interacting with both poly- and monoubiquitin. Overexpression of an Uba domain deletion mutant of ACK1 blocked the ligand-dependent degradation of EGFR, suggesting that ACK1 regulates EGFR degradation via its Uba domain. Thus, ACK1 senses EGF signaling and regulates degradation of EGFR.EGF-induced degradation of EGFR is mediated by ubiquitination (16). The ubiquitination of EGFR is activated upon EGF stimulation by recruiting the RING family E3 ubiquitin ligase Cbl to pY1045 (20, 21). This ubiquitination functions as a sorting signal for transporting EGFR to lysosomes for degradation (14). Nedd4, the HECT domain-containing E3 ubiquitin ligase, is also involved in the regulation of EGFR trafficking by ubiquitination of endocytic or vesicle sorting proteins (28). For example, it has been observed that Nedd4 ubiquitinates Cbl, Eps15, Tsg101, Hrs, and secretory carrier membrane proteins (SCAMPs) and participates in the processes of EGFR endocytosis and degradation (1, 18, 25, 42). However, exactly how Nedd4 engages in the EGFR degradation process in response to EGF stimulation is not known.In this report, we show that EGF stimulation induces ACK degradation. This degradation is associated with ubiquitination of ACK. Nedd4-1, but not Nedd4-2, is identified as the E3 ubiquitin ligase for ubiquitination of ACK. Furthermore, EGF-induced degradation of ACK is EGFR activation dependent and processed by lysosomes. RNAi knockdown and mutational analysis demonstrated that Nedd4-1 and Nedd4-1-catalyzed ubiquitination of ACK are required for EGF-induced degradation of EGFR and ACK. Our findings suggest a new mechanism in regulation of EGFR degradation.     

Calcium-Independent Inhibition of PCSK9 by Affinity-Improved Variants of the LDL Receptor EGF(A) Domain

LDL (low-density lipoprotein) receptor (LDLR) binds to its negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9) through the first EGF (epidermal growth factor-like) domain [EGF(A)]. The isolated EGF(A) domain is a poor antagonist due to its low affinity for PCSK9. To improve binding affinity, we used a phage display approach by randomizing seven PCSK9 contact residues of EGF(A), including the Ca(2+)-coordinating Asp310. The library was panned in Ca(2+)-free solution, and 26 unique clones that bind to PCSK9 were identified. Four selected variants demonstrated improved inhibitory activities in a PCSK9-LDLR competition binding ELISA. The Fc fusion protein of variant EGF66 bound to PCSK9 with a K(d) value of 71nM versus 935nM of wild type [EGF(A)-Fc] and showed significantly improved potency in inhibiting LDLR degradation in vitro and in vivo. The five mutations in EGF66 could be modeled in the EGF(A) structure without perturbation of the EGF domain fold, and their contribution to affinity improvement could be rationalized. The most intriguing change was the substitution of the Ca(2+)-coordinating Asp310 by a Lys residue, whose side-chain amine may have functionally replaced Ca(2+). EGF66-Fc and other EGF variants having the Asp310Lys change bound to PCSK9 in a Ca(2+)-independent fashion. The findings indicate that randomization of an important Ca(2+)-chelating residue in conjunction with "selection pressure" applied by Ca(2+)-free phage selection conditions can yield variants with an alternatively stabilized Ca(2+) loop and with increased binding affinities. This approach may provide a new paradigm for the use of diversity libraries to improve affinities of members of the Ca(2+)-binding EGF domain subfamily.     

Modified Epidermal Growth Factor Receptor (EGFR)-Bearing Liposomes (MRBLs) Are Sensitive to EGF in Solution

Cancers often overexpress EGF and other growth factors to promote cell replication and migration. Previous work has not produced targeted drug carriers sensitive to abnormal amounts of growth factors. This work demonstrates that liposomes bearing EGF receptors covalently crosslinked to p-toluic acid or methyl-PEO₄-NHS ester (or, in short, MRBLs) exhibit an increased rate of release of encapsulated drug compounds when EGF is present in solution. Furthermore, the modified EGF receptors retain the abilities to form dimers in the presence of EGF and bind specifically to EGF. These results demonstrate that MRBLs are sensitive to EGF in solution and indicate that MRBL-reconstituted modified EGF receptors, in the presence of EGF in solution, form dimers which increase MRBL permeability to encapsulated compounds.     

ATG4B (Autophagin-1) Phosphorylation Modulates Autophagy

Autophagy is a catabolic cellular mechanism for entrapping cellular macromolecules and organelles in intracellular vesicles and degrading their contents by fusion with lysosomes. Important roles for autophagy have been elucidated for cell survival during nutrient insufficiency, eradication of intracellular pathogens, and counteracting aging through clearance of senescent proteins and mitochondria. Autophagic vesicles become decorated with LC3, a protein that mediates their fusion with lysosomes. LC3 is a substrate of the cysteine protease ATG4B (Autophagin-1), where cleavage generates a C-terminal glycine required for LC3 conjugation to lipids in autophagosomes. ATG4B both cleaves pro-LC3 and also hydrolyzes lipids from cleaved LC3. We show here that phosphorylation of ATG4B at Ser-383 and Ser-392 increases its hydrolyase activity as measured using LC3 as a substrate. Reconstituting atg4b^−/− cells with phosphorylation-deficient ATG4B showed a role of ATG4B phosphorylation in LC3 delipidation and autophagic flux, thus demonstrating that the cellular activity of ATG4B is modulated by phosphorylation. Proteolytic conversion of pro-LC3 to LC3-I was not significantly impacted by ATG4B phosphorylation in cells. Phosphorylation-deficient ATG4B also showed reduced interactions with the lipid-conjugated LC3 but not unconjugated LC3. Taken together, these findings demonstrate a role for Ser-383 and Ser-392 phosphorylation of ATG4B in control of autophagy.     

Interleukin 17 Receptor A Modulates Monocyte Subsets and Macrophage Generation In Vivo

Interleukin (IL)-17A signaling via Interleukin 17 receptor A (Il17ra) contributes to the inflammatory host response by inducing recruitment of innate immune cells, but also plays a role in homeostatic neutrophilic granulocyte regulation. Monocytes, the other main innate immune cell, have a longer life span and can pursue multiple differentiation pathways towards tissue macrophages. Monocytes are divided into two subpopulations by expression of the Ly6C/Gr1 surface marker in mice. We here investigated the role of Il17ra in monocyte homeostasis and macrophage generation. In Il17ra^-/- and in mixed bone marrow chimeric wt/Il17ra^-/- mice, the concentrations of circulating Il17ra^-/-Gr1^low monocytes were significantly decreased compared to wt cells. Pulmonary, splenic and resident peritoneal Il17ra^-/- macrophages were significantly fewer than of wt origin. Bone marrow progenitor and monocyte numbers were equal, but the proportion of Il17ra^-/-Gr1^low monocytes was already decreased at bone marrow level. After monocyte depletion, initial Gr1^high and Gr1^low monocyte regeneration of Il17ra^-/- and wt cells was very similar. However, Il17ra^-/-Gr1^low counts were not sustained. After labeling with either fluorescent beads or BrdU, Il17ra^-/-Gr1^high monocyte transition to Gr1^low cells was not detectable unlike wt cells. Monocyte recruitment in acute peritonitis, which is known to be largely due to Gr1^high cell migration, was unaffected in an identical environment. Unilateral ureteral obstruction induces a less acute inflammatory and fibrotic kidney injury. Compared to wt cells in the same environment, Il17ra^-/- macrophage accumulation in the kidney was decreased. In the absence of Il17ra on all myeloid cells, renal fibrosis was significantly attenuated. Our data show that Il17ra modulates Gr1^low monocyte counts and suggest defective Gr1^high to Gr1^low monocyte transition as an underlying mechanism. Lack of Il17ra altered homeostatic tissue macrophage formation and diminished renal inflammation and fibrosis. Il17ra appears to be a novel modulator of monocyte phenotype and possible therapeutic target in renal fibrosis.     

Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

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The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

PTK6 Inhibits Down-regulation of EGF Receptor through Phosphorylation of ARAP1

PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin δ-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg¹⁰⁵ residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr²³¹ in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.