Quantitative Proteomics with siRNA Screening Identifies Novel Mechanisms of Trastuzumab Resistance in HER2 Amplified Breast Cancers

HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO₂ chromatography utilizing a dual trap configuration, ∼1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2+ breast cancer patients who develop trastuzumab resistance.HER2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases. Under normal physiologic conditions, HER2 tyrosine kinase signaling is tightly regulated spatially and temporally by the requirement for it to heterodimerize with a ligand bound family member, such as EGFR, HER3/ErbB3, or HER4/ErbB4 (1). However, in 20% to 30% of human breast cancer cases, HER2 gene amplification is present, resulting in a high level of HER2 protein overexpression and unregulated, constitutive HER2 tyrosine kinase signaling (2, 3). HER2 gene amplified breast cancer, also termed HER2-positive breast cancer, carries a poor prognosis, but the development of the HER2 targeted monoclonal antibody trastuzumab (Herceptin) has significantly improved patient survival (2). Despite the clinical effectiveness of trastuzumab, the development of drug resistance significantly increases the risk of patient death. This poses a major medical problem, as most metastatic HER2-positive breast cancer patients develop trastuzumab resistance over the course of their cancer treatment (4). The treatment approach for HER2+ breast cancer patients after trastuzumab resistance develops is mostly a trial-and-error process that subjects the patient to increased toxicity. Therefore, there is a substantial medical need for strategies to overcome trastuzumab resistance.Multiple trastuzumab-resistance mechanisms have been identified, and they alter signaling networks and protein phosphorylation patterns in either a direct or an indirect manner. These mechanisms can be grouped into three categories. The first category is the activation of a parallel signaling network by other tyrosine kinases. These kinases include the receptor tyrosine kinases, EGFR, IGF1R, Her3, Met, EphA2, and Axl, as well as the erythropoietin-receptor-mediated activation of the cytoplasmic tyrosine kinases Jak2 and Src (5–11). The second category is the activation of downstream signaling proteins. Multiple studies have demonstrated activation of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in trastuzumab resistance, which occurs either via deletion of the PTEN lipid phosphatase or mutation of the PI3K genes (12, 13). Activation of Src family kinases or overexpression of cyclin E, which increases the cyclin E–cyclin-dependent kinase 2 signaling pathway, has also been reported (14). The third category includes mechanisms that maintain HER2 signaling even in the presence of trastuzumab. The production of a truncated isoform of HER2, p95HER2, which lacks the trastuzumab binding site, causes constitutive HER2 signaling (15, 16). Overexpression of the MUC4 sialomucin complex inhibits trastuzumab binding to HER2 and thereby maintains HER2 signaling (17, 18).Given that multiple trastuzumab-resistance mechanisms alter signaling networks and protein phosphorylation patterns, we reasoned that mapping phosphotyrosine signaling networks using quantitative proteomics would be a highly useful strategy for analyzing known mechanisms and identifying novel mechanisms of trastuzumab resistance. Quantitative proteomics and phosphotyrosine enrichment approaches have been extensively used to study the EGFR signal networks (19–23). We and others have used these approaches to map the HER2 signaling network (22, 24, 25). Multiple other tyrosine kinase signaling networks were analyzed using quantitative proteomics, including Ephrin receptor, EphB2 (26–28), platelet-derived growth factor receptor (PDGFR) (21), insulin receptor (29, 30), and the receptor for hepatocyte growth factor, c-MET (31).The goal of this study is to identify, quantify, and functionally screen proteins that might be involved in trastuzumab resistance. We used two pairs of HER2 gene amplified trastuzumab-sensitive (parental, SkBr3 and BT474) and -resistant (SkBr3^R and BT474^R) human breast cancer cell lines as models for trastuzumab resistance. These cell lines and their trastuzumab-resistant derivatives have been extensively characterized and highly cited in the breast cancer literature (32, 33). Using stable isotope labeling of amino acids in cell culture (SILAC),¹ phosphotyrosine immunoprecipitations, and online TiO₂ chromatography with dual trap configuration, we quantified the changes in phosphotyrosine containing proteins and interactors between trastuzumab-sensitive and -resistant cells. Several of the known trastuzumab-resistance mechanisms were identified, which serves as a positive control and validation of our approach, and large protein ratio changes were measured in proteins that had not been previously connected with trastuzumab resistance. We then performed a focused siRNA screen targeting the proteins with significantly increased protein ratios. This screen functionally tested the role of the identified proteins and identifies which proteins might have the largest effect on reversing trastuzumab resistance.     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Quantitative Proteomics Discloses MET Expression in Mitochondria as a Direct Target of MET Kinase Inhibitor in Cancer Cells

Cancer cells with MET overexpression are paradoxically more sensitive to MET inhibition than cells with baseline MET expression. The underlying molecular mechanisms are incompletely understood. Here, we have traced early responses of SNU5, a MET-overexpressing gastric cancer cell line, exposed to sublethal concentration of PHA-665752, a selective MET inhibitor, using iTRAQ-based quantitative proteomics. More than 1900 proteins were quantified, of which >800 proteins were quantified with at least five peptides. Proteins whose expression was perturbed by PHA-665752 included oxidoreductases, transfer/carrier proteins, and signaling proteins. Strikingly, 38% of proteins whose expression was confidently assessed to be perturbed by MET inhibition were mitochondrial proteins. Upon MET inhibition by a sublethal concentration of PHA-665752, mitochondrial membrane potential increased and mitochondrial permeability transition pore was inhibited concomitant with widespread changes in mitochondrial protein expression. We also showed the presence of highly activated MET in mitochondria, and striking suppression of MET activation by 50 nm PHA-665752. Taken together, our data indicate that mitochondria are a direct target of MET kinase inhibition, in addition to plasma membrane MET. Effects on activated MET in the mitochondria of cancer cells that are sensitive to MET inhibition might constitute a novel and critical noncanonical mechanism for the efficacy of MET-targeted therapeutics.Recent improvements in survival of some malignancies owe much to advances in uncovering aberrantly active molecular pathways, against which molecularly targeted agents have been developed as new strategies to control cancers (1, 2). However, molecular mechanisms underlying the curious dependence of some cancer cells, which contain multiple genomic, genetic, and epigenetic abnormalities, on a single oncogenic molecule (the phenomenon of “oncogene addiction”) are incompletely understood (3–5).Receptor tyrosine kinases are the most extensively studied oncogenic targets and receptor tyrosine kinase inhibitors have proven anticancer therapeutic efficacy. A receptor tyrosine kinase, MET, whose ligand is hepatocyte growth factor (HGF), is frequently amplified and overexpressed (6, 7) in gastric cancer, the second highest cause of cancer mortality globally (8, 9). Human gastric cancer cell lines harboring MET amplicons and overexpressing MET are readily induced to apoptosis by selective inhibitors of MET (10, 11), several of which are under active development for clinical use (12). One of the selective small molecular inhibitors, PHA-665752, designed chemically as (3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one (molecule weight of 641.61), specifically suppresses tyrosine phosphorylation of MET. PHA-665752 has >50-fold higher selectivity for MET than for other tyrosine and serine/threonine kinases (13). The inhibition of MET kinase function by PHA-665752 on cancer cells had been confirmed with siRNA knockdown of MET, and a number of downstream effectors of MET signaling pathways were confirmed to be effectively abrogated by this compound (10, 13). PHA-665752 has been widely used as a potent and selective tool for the evaluation of MET-dependent cellular functions and signal transduction (10, 14–23).The fact that only a subset of cancers is sensitive to killing by MET-directed therapeutics (hereafter referred to as sensitive cells) (12), raises an unexplained paradox. MET-overexpressing cancer cells could reasonably be expected to be more tolerant of MET kinase inhibition compared with cancer cells that do not overexpress MET. In reality, the opposite occurs. The underlying molecular mechanisms are incompletely understood.To investigate this paradox we undertook a systematic exploration of responses of a MET-overexpressing gastric cancer cell line, SNU5, to sublethal MET inhibition using the iTRAQ-based quantitative proteomics approach. Our results unexpectedly showed a predominant perturbation of mitochondrial proteins in response to MET inhibition. Next, we found that MET inhibition was rapidly associated with altered mitochondrial functions. These observations raised the possibility that mitochondria might be a direct target of MET inhibition. Both protein immunoblotting and confocal microscopy showed the presence of highly activated MET in the mitochondria of sensitive cancer cells. Furthermore, we observed that activating phosphorylation of tyrosine residues of mitochondrial MET was critically modulated by sublethal PHA-665752 treatment.     

Chemokine (C-C Motif) Ligand 2 Engages CCR2+ Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone

Metastatic spread of cancer to distant vital organs, including lung and bone, is the overwhelming cause of breast cancer mortality and morbidity. Effective treatment of systemic metastasis relies on the identification and functional characterization of metastasis mediators to multiple organs. Overexpression of the chemokine (C-C motif) ligand 2 (CCL2) is frequently associated with advanced tumor stage and metastatic relapse in breast cancer. However, the functional mechanism of CCL2 in promoting organ-specific metastasis of breast cancer has not been rigorously investigated. Here, we used organ-specific metastatic sublines of the MDA-MB-231 human breast cancer cell line to demonstrate that overexpression of CCL2 promotes breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 function with a neutralizing antibody reduced lung and bone metastases. The enhancement of lung and bone metastases by CCL2 was associated with increased macrophage infiltration and osteoclast differentiation, respectively. By performing functional assays with primary cells isolated from the wild type, CCL2 and CCR2 knock-out mice, we showed that tumor cell-derived CCL2 depends on its receptor CCR2 (chemokine, CC motif, receptor 2) expressed on stromal cells to exert its function in promoting macrophage recruitment and osteoclast differentiation. Overall, these data demonstrated that CCL2-expressing breast tumor cells engage CCR2⁺ stromal cells of monocytic origin, including macrophages and preosteoclasts, to facilitate colonization in lung and bone. Therefore, CCL2 and CCR2 are promising therapeutic targets for simultaneously inhibiting lung and bone metastasis of breast cancer.Breast cancer is the most common malignancy in women in the United States, with an estimated 182,000 new cases and 40,000 deaths in 2008 (1). Late stage breast cancer patients develop metastases in bone, lung, liver, brain, and other organs, which are responsible for most breast cancer-related mortality and morbidity (2). Severe complications from bone metastasis include debilitating bone fractures, nerve compression and bone pain, and hypercalcemia (3–5), whereas lung metastasis is accompanied by cough, bloody sputum, rib cage pain, and, eventually, failure of the respiratory functions (6). Colonization of different secondary organs by breast cancer is believed to be a complex, multigenic process that depends on productive interactions between tumor cells and stromal microenvironments through concerted actions of organ-specific metastasis genes (7, 8). Functional genomic analysis of preclinical models of breast cancer to bone, lung, and brain have identified distinct sets of organ-specific metastasis genes (9–11), providing novel mechanistic insights into key rate-limiting steps of metastasis to different organs. However, as advanced breast cancer patients often suffer from metastases at several secondary organs, identifying genes that are capable of instigating metastasis to multiple sites may provide the ideal targets for therapeutic intervention of systemic metastasis.Chemokines are small (8–14 kDa) proteins classified into four conserved groups (CXC, CC, C, and CX3C) based on the position of the first two cysteines that are adjacent to the amino terminus (12). They are chemotactic cytokines that stimulate directed migration of leukocytes in response to inflammatory signals. Chemokines are also involved in the maintenance of hematopoietic homeostasis, regulation of cell proliferation, tissue morphogenesis, and angiogenesis (13). Chemokines bind to the seven-transmembrane domain receptors to elicit downstream molecular events that coordinate cell movement. Even though chemokines are unlikely to be a contributing factor for tumor initiation, they can have pleiotropic effects on tumor progression (13, 14). Among more than 50 human chemokines, CCL2 is of particular importance. CCL2, also called monocyte chemoattractant protein-1 (MCP-1), is a potent chemoattractant for monocytes, memory T lymphocytes, and natural killer cells (15). It is involved in a number of inflammatory conditions associated with monocyte recruitment, including delayed hypersensitivity reactions, bacterial infection, arthritis, and renal disease (15). The importance of CCL2 in cancer was manifested by its overexpression in a variety of tumor types, including glioma, ovarian, esophagus, lung, breast, and prostate cancers (15–17). In prostate cancer, CCL2 expression levels was associated with advanced pathological stage (16). Importantly, CCL2-neutralizing antibodies inhibit bone resorption in vitro and bone metastasis in vivo (18–20). In lung cancer, serum CCL2 levels were elevated in lung cancer patients with bone metastasis compared with localized diseases. Neutralizing antibodies against CCL2 also inhibited the tumor conditioned media-induced osteoclast formation in vitro and bone metastasis in vivo (17). Taken together, these findings suggested a role of CCL2 in bone metastasis.A potential role of CCL2 in breast cancer progression and metastasis has been indicated by the analysis of CCL2 expression of tumor and serum samples from breast cancer patients. Serum CCL2 levels were significantly higher in postmenopausal breast cancer patients than in age-matched controls (21). Over 50% of breast cancer tumor samples had intense staining of CCL2 in tumor cells (22). Prognostic analysis further revealed that high expression of CCL2 was correlated with advanced tumor stage, lymph node metastasis (23), and early relapse (24). CCL2 up-regulation in breast tumors was also associated with the infiltration of tissue-associated macrophages (TAMs)³ and with increased microvessel density (22, 24). TAMs have been known to contribute to primary tumor progression and metastasis of breast cancer (25), which is supported by epidemiological evidence showing that TAM infiltration portended a poor clinical outcome (26, 27). However, whether the function of CCL2 in modulating activity of macrophages and possibly other cell types is important for breast tumor organotropic metastasis has not been rigorously investigated. CCL2 may engage organ-specific cell types derived from the same bone marrow myelomonocytic progenitors. These progenitors differentiate into osteoclast precursors in bone or into blood monocytes that eventually become mature macrophages in different tissues, like alveolar macrophages in lung (28). These stromal cell types of myelomonocytic origin may contribute to different functions in different organ-specific metastases. Another unresolved question regarding the function of CCL2 in tumor-stroma interaction is the functional involvement of CCL2 receptors. CCL2 can bind to both CCR2 and CCR4 (29, 30). Loss of function studies in mice showed CCL2 and CCR2 knock-out mice displayed similar impairments in monocyte migration (31, 32), suggesting that CCR2 is the major functional receptor for CCL2. Understanding whether CCR2 deficiency in stromal cells leads to compromised monocyte engagement by CCL2-expressing tumor cells may have important implications in designing targeting therapeutics against the CCL2/CCR2 axis.In this study, we used the recently developed organ-specific metastatic sublines of the human breast cancer cell MDA-MB-231 (9, 10, 33) and showed that overexpression of CCL2 promotes both lung and bone metastases. This function was associated with increased TAM infiltration in lung metastasis and increased osteoclast differentiation in bone metastasis, respectively. Furthermore, by using macrophages and bone marrow cells isolated from wild type, CCL2-deficient, and CCR2-deficient mice, we showed that CCR2 expression in stromal cells is essential for tumor-derived CCL2 to recruit macrophages and promote osteoclastogenesis. Targeting tumor-derived CCL2 by a neutralizing antibody significantly reduced metastasis formation in both bone and lung.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Multipattern Consensus Regions in Multiple Aligned Protein Sequences and Their Segmentation

Decomposing a biological sequence into its functional regions is an important prerequisite to understand the molecule. Using the multiple alignments of the sequences, we evaluate a segmentation based on the type of statistical variation pattern from each of the aligned sites. To describe such a more general pattern, we introduce multipattern consensus regions as segmented regions based on conserved as well as interdependent patterns. Thus the proposed consensus region considers patterns that are statistically significant and extends a local neighborhood. To show its relevance in protein sequence analysis, a cancer suppressor gene called p53 is examined. The results show significant associations between the detected regions and tendency of mutations, location on the 3D structure, and cancer hereditable factors that can be inferred from human twin studies.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]     

Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Phosphoproteomics of Klebsiella pneumoniae NTUH-K2044 Reveals a Tight Link between Tyrosine Phosphorylation and Virulence

Encapsulated Klebsiella pneumoniae is the predominant causative agent of pyogenic liver abscess, an emerging infectious disease that often complicates metastatic meningitis or endophthalmitis. The capsular polysaccharide on K. pneumoniae surface was determined as the key to virulence. Although the regulation of capsular polysaccharide biosynthesis is largely unclear, it was found that protein-tyrosine kinases and phosphatases are involved. Therefore, the identification and characterization of such kinases, phosphatases, and their substrates would advance our knowledge of the underlying mechanism in capsule formation and could contribute to the development of new therapeutic strategies. Here, we analyzed the phosphoproteome of K. pneumoniae NTUH-K2044 with a shotgun approach and identified 117 unique phosphopeptides along with 93 in vivo phosphorylated sites corresponding to 81 proteins. Interestingly, three of the identified tyrosine phosphorylated proteins, namely protein-tyrosine kinase (Wzc), phosphomannomutase (ManB), and undecaprenyl-phosphate glycosyltransferase (WcaJ), were found to be distributed in the cps locus and thus were speculated to be involved in the converging signal transduction of capsule biosynthesis. Consequently, we decided to focus on the lesser studied ManB and WcaJ for mutation analysis. The capsular polysaccharides of WcaJ mutant (WcaJY5F) were dramatically reduced quantitatively, and the LD₅₀ increased by 200-fold in a mouse peritonitis model compared with the wild-type strain. However, the capsular polysaccharides of ManB mutant (ManBY26F) showed no difference in quantity, and the LD₅₀ increased by merely 6-fold in mice test. Our study provided a clear trend that WcaJ tyrosine phosphorylation can regulate the biosynthesis of capsular polysaccharides and result in the pathogenicity of K. pneumoniae NTUH-K2044.Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications in both eukaryotic and prokaryotic organisms. It is best known that protein phosphorylation is a reversible enzyme-catalyzed process that is controlled by various kinases and phosphatases. The aberrant functions often result in irregular protein phosphorylation and ultimately lead to serious disease states such as malignant transformation, immune disorders, and pathogenic infections in mammals (1, 2). Recently, accumulating evidences suggest that Ser/Thr/Tyr phosphorylations also contribute to regulate a diverse range of cellular responses and physiological processes in prokaryotes (1). Among them, tyrosine phosphorylation in encapsulated bacteria has been discovered to play key roles in capsular polysaccharide (CPS1; K antigen) biosynthesis, which leads to virulence (3, 4). This thick layer of exopolysaccharide on many pathogenic bacteria can act as a physical boundary to evade phagocytosis and complement-mediated killing and further inhibit complement activation of the host (1, 5, 6).In 1996, Acinetobacter johnsonii protein-tyrosine kinase (Ptk) was first discovered and categorized under the bacterial protein-tyrosine kinase (BY-kinase) family (1, 7, 8). Shortly after, its function in bacterial exopolysaccharide production and transport was characterized (1, 7, 8). From then on, many more bacterial tyrosine kinases such as Wzc of Escherichia coli (1, 9) and EpsB of Pseudomonas solanacearum (10, 11) were found to possess this conserved property; deletion of such tyrosine kinases will result in the loss of exopolysaccharide production (12). Therefore, several experiments were conducted to investigate the role of the downstream substrates of the tyrosine kinases in different strains of bacteria, and some targeted proteins were found to participate in the exopolysaccharide anabolism (13, 14). These findings demonstrated a direct relationship between bacterial tyrosine phosphorylation and exopolysaccharide biosynthesis that was directly reflected in the strain virulence.In the past, the functional roles of the critical components involved in protein phosphorylation were defined by basic biochemical and genetic approaches (1). However, there exists a salient gap between the growing number of identified protein-tyrosine kinases/phosphatases and the relative paucity of protein substrates characterized to date. Genomic sequence analyses and advanced high resolution/high accuracy MS systems with vastly improved phosphopeptide enrichment strategies are among the two key enabling technologies that allow a high efficiency identification of the scarcely detectable site-specific phosphorylations in bacterial systems (15). Mann et al. (16) were the first to initiate a systematic study of the phosphoproteome of B. subtilis in 2007 followed by similar site-specific phosphoproteomics analyses of E. coli (17), Lactococcus lactis (18), and Halobacterium salinarum (19). These pioneering works have since set the foundation in bacterial phosphoproteomics but have not been specifically carried out to address a particular biological issue of causal relevance to virulence or pathogenesis.Klebsiella pneumoniae is a Gram-negative, non-motile, facultative anaerobic, and rod-shaped bacterium. It is commonly found in water and soil (20) as well as on plants (21) and mucosal surfaces of mammals, such as human, horse, and swine (22, 23). It was demonstrated that CPS on the surface of K. pneumoniae is the prime factor of virulence and toxicity in causing pyogenic liver abscess (PLA), a common intra-abdominal infection with a high 10–30% mortality rate worldwide (24–29). There are also variations in virulence in regard to different capsular serotypes; K1 and K2 were found to be especially pathogenic in causing PLA in a mouse model (30) compared with other serotypes, which show little or no effect (31–34). The K. pneumoniae NTUH-K2044 (K2044) strain, encapsulated with K1 antigen (35), was isolated from clinical K. pneumoniae liver abscess patients. It has become an important emerging pathogen (36) because it usually complicates metastatic septic endophthalmitis and irreversible central nervous system infections independent of host underlying diseases (30, 34). The transmission rate is high (37), and it often rapidly leads to outbreaks of community-acquired infections, such as bacteremia, nosocomial pneumonia, and sepsis, common in immunocompromised individuals (38).In this study, we wanted to prove that the biosynthesis of CPS is mediated through tyrosine phosphorylation of a subset of proteins. An MS-based systematic phosphoproteomics analysis was conducted on K2044 to identify tyrosine phosphorylated proteins that are also associated with CPS biosynthesis. We further validated the relationship between tyrosine phosphorylation on those proteins and virulence of K2044 by site-directed mutagenesis, CPS quantification, serum killing, and mouse lethality assay.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.