Potential Tyrosine Phosphorylation Sites Research Articles

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

The Diverse Role of CUB and Sushi Multiple Domains 1 (CSMD1) in Human Diseases.

CUB and Sushi Multiple Domains 1 (CSMD1), a tumour suppressor gene, encodes a large membrane-bound protein including a single transmembrane domain. This transmembrane region has a potential tyrosine phosphorylation site, suggesting that CSMD1 is involved in controlling cellular functions. Although the specific mechanisms of action for CSMD1 have not yet been uncovered, it has been linked to a number of processes including development, complement control, neurodevelopment, and cancer progression. In this review, we summarise CSMD1 functions in the cellular processes involved in the complement system, metastasis, and Epithelial mesenchymal transition (EMT) and also in the diseases schizophrenia, Parkinson's disease, and cancer. Clarifying the association between CSMD1 and the aforementioned diseases will contribute to the development of new diagnosis and treatment methods for these diseases. Recent studies in certain cancer types, e.g., gastric cancer, oesophageal cancer, and head and neck squamous cell carcinomas, have indicated the involvement of CSMD1 in response to immunotherapy.

Abstract 2000: Cancer metastasis driven by the novel MEK1 substrate MACC1 is restricted by clinically applicable MEK1 inhibitors

Abstract Cancer metastasis is responsible for >90% of cancer deaths and remains a major treatment challenge. In search of new drivers of metastasis we identified the novel, previously undescribed gene Metastasis-Associated in Colon Cancer 1 (MACC1) in human colorectal cancer (CRC). MACC1 induces fundamental processes like proliferation, migration, invasiveness and metastasis in xenografted and transgenic mice. MACC1 has been established by us and many other groups as key player, prognostic and predictive biomarker for tumor progression and metastasis in >20 solid cancer types. Proof-of-concept for MACC1 as a therapeutic target to restrict cancer progression and metastasis was provided by transcriptional and post-transcriptional downregulation of MACC1 for several solid cancers, including CRC. Specific inhibitors targeting MACC1 post-translational protein modifications to restrict tumor growth and metastasis are not identified so far. Here we report the identification of MACC1 as a newly identified substrate of the kinase MEK1 (MAP2K1). MEK1 directly phosphorylates MACC1 leading to accelerated and increased ERK1 activation. Mutating three potential hierarchical MACC1 tyrosine phosphorylation sites abrogates MACC1 dependent target gene expression like MET, cell proliferation and motility in cell culture and importantly, metastasis in mouse xenograft models. Targeting MEK1 by RNAi or by clinically applicable MEK1 inhibitors like AZD6244 and GSK1120212 reduces MACC1 tyrosine phosphorylation and restricts MACC1-induced metastasis formation. Our findings demonstrate that MAP kinase signaling is not linearly leading only to ERK activation, but branches at the level of MEK1. In summary, MACC1 tyrosine phosphorylation is decisive for tumor growth and metastasis. The fundamental finding of MACC1 being a newly identified MEK1 substrate opens new therapeutic options with potential for clinical translation. Targeting MACC1 tyrosine phosphorylation using MEK1 inhibitors thereby intervening in MACC1-induced metastasis aims at the ultimate goal of personalized therapies for inhibition of cancer progression and metastasis, resulting in improved patient survival. Since MACC1 is confirmed as decisive driver for tumor growth and metastasis in a variety of solid cancers, the findings made here for CRC might be translated to further solid tumor types. The usefulness of MACC1 as therapeutic target towards MEK1 inhibitor treatment requires confirmation in clinical trials. Citation Format: Dennis Kobelt, Daniel Perez-Hernandez, Claudia Fleuter, Mathias Dahlmann, Fabian Zincke, Janice Smith, Rebekka Migotti, Susen Burock, Wolfgang Walther, Gunnar Dittmar, Ulrike S. Stein. Cancer metastasis driven by the novel MEK1 substrate MACC1 is restricted by clinically applicable MEK1 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2000.

Specific dephosphorylation at tyr-554 of git1 by ptprz promotes its association with paxillin and hic-5.

G protein-coupled receptor kinase-interactor 1 (Git1) is involved in cell motility control by serving as an adaptor that links signaling proteins such as Pix and PAK to focal adhesion proteins. We previously demonstrated that Git1 was a multiply tyrosine-phosphorylated protein, its primary phosphorylation site was Tyr-554 in the vicinity of the focal adhesion targeting-homology (FAH) domain, and this site was selectively dephosphorylated by protein tyrosine phosphatase receptor type Z (Ptprz). In the present study, we showed that Tyr-554 phosphorylation reduced the association of Git1 with the FAH-domain-binding proteins, paxillin and Hic-5, based on immunoprecipitation experiments using the Tyr-554 mutants of Git1. The Tyr-554 phosphorylation of Git1 was higher, and its binding to paxillin was consistently lower in the brains of Ptprz-deficient mice than in those of wild-type mice. We then investigated the role of Tyr-554 phosphorylation in cell motility control using three different methods: random cell motility, wound healing, and Boyden chamber assays. The shRNA-mediated knockdown of endogenous Git1 impaired cell motility in A7r5 smooth muscle cells. The motility defect was rescued by the exogenous expression of wild-type Git1 and a Git1 mutant, which only retained Tyr-554 among the multiple potential tyrosine phosphorylation sites, but not by the Tyr-554 phosphorylation-defective or phosphorylation-state mimic Git1 mutant. Our results suggested that cyclic phosphorylation-dephosphorylation at Tyr-554 of Git1 was crucial for dynamic interactions between Git1 and paxillin/Hic-5 in order to ensure coordinated cell motility.

Abstract 5113: Carboxyl terminal region of MUC16 is critical for tumor cell invasion and drug resistance.

Abstract MUC16, also known as CA125, is routinely used to monitor the progression (as a biomarker) and response to therapeutics (as a prognostic marker) for epithelial ovarian cancer (EOC), but the deregulated expression is not restricted only to EOC. Recent studies from our lab have shown that normal breast and pancreas (ducts) do not express MUC16; however its expression progressively increases in primary and metastatic breast and pancreatic cancers. In addition, we have observed the involvement of MUC16 in enhancing the tumorigenic potential by shRNA studies. In the present study, to understand the mechanistic involvement of MUC16, we have focused on its C-terminal region. MUC16 is a type-I membrane-bound mucin with a heavily glycosylated N-terminal domain, a transmembrane region (TM) and a 32-residue cytoplasmic tail (CT) domain. Though it has been predicted that MUC16 undergoes cleavage/shedding in the extracellular SEA domain(s) by proteases such as MMP7 and neutrophil elastase, the exact nature and site of cleavage and the fate of the membrane bound portion (C-terminal) after release of extracellular portion is not entirely understood. The post cleavage C-ter region is interesting due to presence of (a) stretch of polybasic aminoacids, the site of interaction for cytoskeletal proteins like Ezrin/Radixin/Moesin, (b) potential nuclear localization signal (bioinformatics prediction) which might facilitate its nuclear localization with associated transcription factors modulating the transcription of various target genes and (c) several potential serine, threonine and tyrosine phosphorylation sites facilitating its interaction(s) with proteins influencing oncogenic signaling pathways. Therefore, we hypothesized that the oncogenic potential of MUC16 is in part, mediated by the potential involvement of C-terminal domain of MUC16. Thusly, we have made several truncated versions of MUC16 from the C-terminal end with N-terminal FLAG tag and C-terminal HA-tag and are in the process of characterization. To understand the functional significance of this C-terminal region, we have stably transfected MCF7 (breast cancer), MiaPaCa and T3M4 (pancreatic cancer) cells with an empty vector, FLAG-MUC16-114AA-HA and FLAG-MUC16-114AA-dCT (32 AA in the CT is deleted). Our results from in vitro functional assays indicate that C-terminal region of MUC16 is responsible for enhancing the invasive potential of cancer cells and confers resistance to chemotherapeutic agents such as Cisplatin and Gemcitabine. Moreover, we see that the function of the carboxyl terminal MUC16 is dependent on the stability of the protein, which is a function of N-glycosylation and ubiquitylation. Overall, our studies indicate that C-terminal region of MUC16 is an important mediator of cancer cell invasion and drug resistance. (This work is supported from the Department of Defense BC101014). Citation Format: Srustidhar Das, Maria P. Torres-Gonzalez, Prabin D. Majhi, Imayavaramban Lakshmanan, Moorthy P. Ponnusamy, Satyanarayana Rachagani, Eric Cruz, Dhanya Haridas, Surinder K. Batra. Carboxyl terminal region of MUC16 is critical for tumor cell invasion and drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5113. doi:10.1158/1538-7445.AM2013-5113

Development of unique antibodies directed against each of the six different phosphotyrosine residues within the T cell receptor CD3ζ chain

Signal transduction from the T cell antigen receptor (TCR)/CD3 complex involves six different immunoreceptor tyrosine-based activation motifs (ITAM) located within the cytoplasmic tails of the CD3 chains. Each ITAM possesses two conserved tyrosine residues that can undergo phosphorylation upon TCR/CD3 crosslinking and become a docking site for SH2-containing effector molecules. Specificity of the SH2 domains is determined by their ability to bind a phosphorylated tyrosine in the context of a longer peptide motif within the target protein. As a result, phosphorylation of different tyrosines within the CD3 cytoplasmic tails creates docking sites for distinct SH2-containing signaling proteins that differentially impact on the quality of the T cell response.In the present study, we prepared antibodies specific for each of the six different phosphotyrosines of the mouse CD3ζ chain. The antibodies were characterized with respect to their cross-reactivity, ability to recognize the phosphorylated versus non-phosphorylated forms of tyrosine-containing motifs, and cross-reactivity with the homologous phospho-motifs on the human CD3ζ protein.The antibodies were found to be specific and selective for phospho-CD3ζ. They can serve as useful tools for distinguishing between the six potential tyrosine phosphorylation sites on the CD3ζ chain, and for correlating the phosphorylation of specific CD3ζ tyrosine residues with activation of signaling pathways that dictate T cell differentiation into responding, anergic, or apoptotic cells.

Abstract 4009: Identification of Y726 and Y793 as novel phosphorylation sites in human Flt3 and their role in signaling

Abstract Fms-like tyrosine kinase-3 (FLT3), a growth factor receptor expressed in hematopoietic progenitor cells. FLT3 mutations, ITD and D835, are found in approximately 30% of AML patients and correlate with a poor prognosis, thus making receptor a potential therapeutic target. Flt3 mutations cause constitutive activation of the receptor, leading to alteration in signal transduction. The aims of this study are to analyze the kinetics and specificity of FLT3 autophosphorylation in wild-type FLT3 and FLT3 mutants, to identify new phosphorylation sites in FLT3 receptor and to determine related signaling pathways. Using a panel of “homemade” phosphospecific antibodies directed against potential tyrosine phosphorylation sites in FLT3, we identified three novel phosphorylation sites and studied the kinetics and specificity of phosphorylation in living cells. Our results show that activation of the wild-type Flt3 receptor is ligand- dependent and responding in a time dependent manner in contrast to FLT3-ITD and D835Y that are constitutive active and ligand-independent. Eight phosphorylated tyrosine residues (pY589, pY591, pY599, pY726, pY768, pY793, pY842 and pY955) were shown to have different phosphorylation characteristics in the wild-type receptor compared to the mutated receptors. Furthermore, we showed that Y726, Y793 and Y842 are novel phosphorylation sites of FLT3 receptor. Further study on stable transfected cells with Y-to-F mutations (726F and 793F), showed that these sites might be important for cell survival, proliferation and transformation. We could also see decreased phosphorylation of Erk1/2 and SHP2 in mutants compare to wild-type FLT3 or FLT3-ITD. Potential binding partners for Y726 and Y793 were predicted by using synthetic phosphopeptides for probing of SH2 domain arrays. The candidate binding partners will be verified as true binding partners using co-immunoprecipitation experiments. From this study we could observe not only quantitative changes but also qualitative differences in the phosphorylation patterns of wild-type and mutated FLT3 receptors. We have also identified novel phosphorylation sites in FLT3 receptor that can be involved in different signaling pathways. Using this data we can further study the mechanisms of signaling pathways of the Flt3 receptor that are involved in many biological responses and understand the mechanism by which FLT3-ITD and -D835Y functions in pathological conditions. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4009.

Abstract 5208: PDGFRα stimulates glioma cell invasion through tyrosine phosphorylation of Dock180 at Y1811 and activation of the Dock180-CrkII-p130cas-Rac1 pathway

Abstract Platelet-derived growth factor receptor (PDGFR)α ranks third among the top 11 amplified genes in high-grade glioblastoma multiforme (GBM) and is frequently overexpressed in low-grade gliomas. However, the molecular mechanisms by which PDGFRα promotes glioma growth and invasion are largely unknown. Here we report that PDGFRα induces glioma cell invasion through tyrosine phosphorylation of Dock180, a Rac1 guanine nucleotide exchange factor (GEF). By immunohistochemical analysis of primary glioma tissue specimens, we found that PDGFRα, PDGF-A and Dock180 are co-expressed in invasive areas but not the central regions of the clinical biopsies. Ectopic expression of PDGF-A by human glioma LN444 cells with endogenous PDGFRα significantly promotes glioma growth and invasion in the brain of mice. Cellular depletion of Dock180 by RNAi in LN444 and LN443 cells inhibits PDGF-A-promoted cell migration in vitro and suppressed PDGF-A-expressing LN444 glioma infiltration and growth in the brain. In vitro, PDGF-A stimulates cell migration, invasion and induces tyrosine phosphorylation of Dock180, whereas AG1296, an inhibitor of PDGFRα inhibits the PDGF-A-induced phosphorylation of Dock180 and glioma cell migration. By co-expression of Dock180, its various deletion mutants and an oncogenic mutant PDGFRαΔ8,9, we identified tyrosine residue (Y) 1811 of Dock180 as a major phosphorylation (p-Y) site of Dock180. Y1811 is located within the CrkII-binding domain at the C-terminus of Dock180 and a potential tyrosine phosphorylation site for Src kinase. Y1811 is also highly conserved in Dock180 protein from various species. Mutation of the Y to a phenylalanine (F) at Y1811 of Dock180 significantly attenuates PDGFRα-induced phosphorylation and subsequently decreases Rac1 GTPase activity. Co-expression of Dock180 and PDGFRα and CrkII promotes PDGF-A-induced association of Dock180 with CrkII and p130Cas whereas the Y1811F mutant markedly disrupts their association. Inhibition of Src by its inhibitors and a dominant negative Src mutant attenuates PDGF-A-stimulated p-Y of wild-type Dock180, Rac1 activity and cell migration while a constitutively active Src mutant induces p-Y of wild-type Dock180 but not Dock180 Y1811F mutant. Finally, re-introduction of wild-type Dock180 into LN444/PDGF-A/shRNA-Dock180 cells that endogenous Dock180 was depleted rescued PDGFRα-promoted CrkII association, Rac1 activity and glioma cell motility in vitro, and tumor growth and invasion the brain whereas re-expression of Dock180 Y1811F mutant in these cells failed to restore PDGFRα-stimulated cellular behaviors. Taken together, this data reveals a molecular mechanism by which PDGFRα stimulates glioma cell invasion through tyrosine phosphorylation at Y1811 of a Rac1 GEF, Dock180, leading to increases in Rac1 activity and glioma cell invasion in the brain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5208.

SHIP-1 Associates with Tyrosine Phosphorylated Protein Kinase C-Delta at 155 Residue.

Abstract 3004Poster Board II-981Protein Kinase C-delta (PKCδ) is a novel PKC isoform that differentially regulates platelet dense granule secretion. PKCδ positively regulates Protease activated receptor (PAR)-mediated dense granule secretion, whereas it negatively regulates glycoproteinVI (GPVI)-mediated dense granule secretion in platelets. PKCδ, a serine/threonine kinase is phosphorylated on its tyrosine residues. There are nine potential tyrosine phosphorylation sites in the regulatory domain of PKCδ. Phosphorylation at different tyrosine residues regulates its substrate specificity. We have previously shown that the association of PKCδ with Lyn and SHIP-1 negatively regulates GPVI-mediated dense granule secretion. However, the event leading to the association between PKCδ and SHIP-1 is not known. We hypothesize that the differential tyrosine phosphorylation of PKCδ downstream of PARs or GPVI receptors result in the preferential association with SHIP-1. In the current study, we show that PKCδ is phosphorylated at tyrosine residues Y332, Y523, Y525 and Y565 upon PAR or GPVI stimulation. Y311 residue is predominantly phosphorylated upon stimulation of PARs, whereas Y155 residue is preferentially phosphorylated upon GPVI stimulation. PAR-mediated Y311 phosphorylation peaks at later timepoint, whereas GPVI-mediated Y155 phosphorylation peaks at an early timepoint. correlating with dense granule secretion. Furthermore, we show that agarose-conjugated Y155 phosphorylated PKCδ peptide associates with SHIP-1 upon GPVI stimulation, and not PARs. These data suggest that the phosphorylation of PKCδ at distinct tyrosine residues differentially regulate its association with SHIP-1. Therefore, we conclude that the GPVI-mediated phosphorylation of PKCδ at 155 is required for its association with SHIP-1.This study is supported by pre-doctoral fellowship to Ramya Chari from American Heart Association, Pennsylvania-Delaware affiliate. Disclosures:No relevant conflicts of interest to declare.

Oncogenic Flt3 receptors display different specificity and kinetics of autophosphorylation

Fms-like tyrosine kinase-3 (Flt3), a growth factor receptor normally expressed in hematopoietic progenitor cells, has been shown to have an important role in development of acute myeloid leukemia (AML) due to activating mutations. Flt3 mutations are found in approximately one-third of AML patients and correlate with a poor prognosis, thus making the Flt3 receptor a potential therapeutic target. The aim of the investigation was to analyze the kinetics and specificity of Flt3 autophosphorylation in wild-type Flt3 as well as in oncogenic Flt3 mutants. We have used Ba/F3 cells stably expressing either wild-type, internal tandem duplication, or D835Y mutants of Flt3 in order to compare the site selectivity of tyrosine phosphorylation sites. By the use of a panel of phosphospecific antibodies directed against potential tyrosine phosphorylation sites in Flt3, we identified several novel phosphorylation sites in Flt3 and studied the kinetics and specificity of ligand-induced phosphorylation in living cells. Eight phosphorylated tyrosines (pY589, pY591, pY599, pY726, pY768, pY793, pY842, and pY955) were investigated and shown to be differentially phosphorylated in the wild-type versus the mutated receptors. Furthermore, we show that tyrosines 726, 793, and 842 are novel phosphorylation sites of Flt3 in intact cells. In this study, we have looked at the site-specific phosphorylation in the wild-type Flt3 in comparison to the mutants found in AML. We observed not only quantitative changes but, more importantly, qualitative differences in the phosphorylation patterns of the wild-type and the mutated Flt3 receptors, which might enhance the understanding of the mechanisms by which Flt3 contributes to AML in patients with mutations in Flt3.

Systematic Mutagenesis of the Murine Gammaherpesvirus 68 M2 Protein Identifies Domains Important for Chronic Infection

Murine gammaherpesvirus 68 (MHV68) infection of inbred mice represents a genetically tractable small-animal model for assessing the requirements for the establishment of latency, as well as reactivation from latency, within the lymphoid compartment. By day 16 postinfection, MHV68 latency in the spleen is found in B cells, dendritic cells, and macrophages. However, as with Epstein-Barr virus, by 3 months postinfection MHV68 latency is predominantly found in isotype-switched memory B cells. The MHV68 M2 gene product is a latency-associated antigen with no discernible homology to any known cellular or viral proteins. However, depending on experimental conditions, the M2 protein has been shown to play a critical role in both the efficient establishment of latency in splenic B cells and reactivation from latently infected splenic B cells. Inspection of the sequence of the M2 protein reveals several hallmarks of a signaling molecule, including multiple PXXP motifs and two potential tyrosine phosphorylation sites. Here, we report the generation of a panel of recombinant MHV68 viruses harboring mutations in the M2 gene that disrupt putative functional motifs. Subsequent analyses of the panel of M2 mutant viruses revealed a functionally important cluster of PXXP motifs in the C-terminal region of M2, which have previously been implicated in binding Vav proteins (P. A. Madureira, P. Matos, I. Soeiro, L. K. Dixon, J. P. Simas, and E. W. Lam, J. Biol. Chem. 280:37310-37318, 2005; L. Rodrigues, M. Pires de Miranda, M. J. Caloca, X. R. Bustelo, and J. P. Simas, J. Virol. 80:6123-6135, 2006). Further characterization of two adjacent PXXP motifs in the C terminus of the M2 protein revealed differences in the functions of these domains in M2-driven expansion of primary murine B cells in culture. Finally, we show that tyrosine residues 120 and 129 play a critical role in both the establishment of splenic latency and reactivation from latency upon explant of splenocytes into tissue culture. Taken together, these analyses will aide future studies for identifying M2 interacting partners and B-cell signaling pathways that are manipulated by the M2 protein.

Radial spoke protein 44 (human meichroacidin) is an axonemal alloantigen of sperm and cilia

To identify novel sperm alloantigens relevant to immune infertility, sera from infertile men containing antisperm antibodies (ASA) were employed on 2-D immunoblots of human sperm proteins. An immunoreactive protein spot (MW: 44 kDa, pI: 4.5) was microsequenced and the related cDNA was cloned yielding a 309 amino acid sequence corresponding to a gene currently annotated in Genbank as TSGA2 homolog (mouse) to signify ‘testis specific gene A2’. In Genbank the protein deduced from this gene is currently named human meichroacidin, an orthologue of meichroacidin previously identified in mouse spermatocytes. Human TSGA2 mapped to chromosome 21q22.3. Human meichroacidin (hMCA) contained a single potential tyrosine phosphorylation site and five casein kinase phosporylation motifs. The N-terminus contained a Membrane Occupation Recognition Nexus (MORN) motif found in the lipid kinase-phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family and junctophilins. However hMCA lacked the characteristic kinase homology domain of PIP5K. Northern blot analysis revealed 1.5 kb hMCA transcripts in testis and trachea with lower levels in thyroid and spinal cord. A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis demonstrated occurrence of the mRNA messages in all the ciliated tissues tested with highest levels of messages in testis and trachea. Western blot analysis showed the presence of hMCA protein in brain, thyroid and trachea at the identical mass, 44 kDa, as in human testis. However, this immunoreactive pattern differed from that of sperm in which a 38 kDa form was also evident suggesting that hMCA undergoes proteolytic processing. In human testis, hMCA localized to the tails of developing spermatids and did not localize to the nucleus of either spermatocytes or spermatids. EM immunocytochemistry localized hMCA within the radial spokes of the axonemal complex of the sperm flagellum, and immunofluorescence studies revealed h-meichroacidin in the cilia of epithelial cells in the trachea and ependyma. Bioinformatic identification of orthologues of meichroacidin in several lower organisms including ciliates and flagellates suggest the protein plays a role in flagellar motility across phyla. We propose the term radial spoke protein 44 as an accurate designation, preferable to human meichroacidin because it denotes the restricted localization of the protein to the radial spokes of the axonemes of both sperm and cilia. Further, since the human gene is expressed in brain, thyroid, trachea and lung in addition to testis, we suggest that the gene name be changed from TSGA2 [testis specific gene A2] to radial spoke protein 44 [ RSP44].

Point Mutation at Single Tyrosine Residue of Novel Oncogene NOK Abrogates Tumorigenesis in Nude Mice

Receptor protein-tyrosine kinases (RPTKs) are tightly regulated during normal cellular processes including cell growth, differentiation, and metabolism. Recently, a RPTK-like molecule named novel oncogene with kinase-domain (NOK) has been cloned and characterized. Overexpression of NOK caused severe cellular transformation as well as tumorigenesis and metastasis in nude mice. In the current study, we generated two tyrosine-->phenylalanine (Y-->F) point mutations (Y327F and Y356F) within the endodomain of NOK that are well conserved in many RPTK subfamilies and are the potential tyrosine phosphorylation sites important for major intracellular signaling. Using BaF3 cells stably expressing the ectodomain of mouse erythropoietin receptor, and the transmembrane and endodomain of NOK (BaF3-E/N), we were able to show that point mutations at either Y327 or Y356 dramatically blocked cellular transformation by NOK as examined by colony formation and cellular DNA synthesis. In addition, tumorigenesis induced by BaF3-E/N was completely abrogated upon the introduction of either single mutation. Importantly, signaling studies revealed that the activation of extracellular signal-regulated kinase was inhibited by Y356F and was significantly reduced by Y327F. Both mutations significantly impaired Akt phosphorylation. Interestingly, both mutations did not affect the kinase activity of NOK. Moreover, apoptotic analysis revealed that both mutations accelerated cell death by activating caspase-3-mediated pathways. Thus, our study shows that these potential tyrosine phosphorylation sites may play critical roles in NOK-mediated tumorigenesis both in vitro and in vivo.

Cloning of two Atlantic salmon growth hormone receptor isoforms and in vitro ligand-binding response

Two isoforms of the full-length cDNA of the growth hormone receptor (GHR) of the Atlantic salmon (Salmo salar; ss) were cloned by a PCR approach using RACE. Respectively, the cDNA sequences of ssGHR isoforms 1 and 2 are 2654 and 2608 nucleotides long, with 1782 and 1773 nucleotide ORFs. The resulting coded proteins are 594 and 590 aa long, with 19 and 20 aa signal peptides. The two isoforms share 86% protein and 87% cDNA sequence similarity. Isoform 1 is most similar to other salmonid GHR isoforms 1 while isoform 2 is most similar to salmonid GHR isoforms 2 (93–5%). Similarity with other teleost species was lower (37–4%). The bioactivity of the cloned ssGHR was tested by transfecting the ssGHR isoform 1 cDNA into CHO-K1 hamster cells, incubating with recombinant salmon GH (sGH) or native ovine prolactin (oPRL), and measuring cell proliferation by the MTT assay. The ssGHR-transfected cells significantly increased proliferation when stimulated by sGH at all concentrations. oPRL stimulated ssGHR-transfected cells at higher concentrations due to receptor cross reaction. ssGHR isoforms 1 and 2 contain a single transmembrane domain and the typical conserved motifs found in other teleost GHRs, including four paired cysteine residues and five potential N-glycosylation sites in the extracellular domain, Box I and Box II, as well as seven potential tyrosine phosphorylation sites in the intracellular domain. However, in salmonids, these motifs differ from those of other teleosts, and could be responsible for differentiated hormone binding, signal transduction and response.

Functional Domains of the Mouse β3-Adrenoceptor Associated with Differential G Protein Coupling

Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.

Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

Signaling by the Granulocyte-Colony Stimulating Factor Receptor Involves Jak2-Dependent Phosphorylation of Gab2.

Granulocyte colony stimulating factor (G-CSF) is the major cytokine involved in neutrophil production. G-CSF has pleiotropic effects on myeloid cells, initially stimulating proliferation but later promoting differentiation. The specific signaling pathways that mediate the diverse effects of G-CSF remain incompletely understood. Recently, the scaffolding molecule Grb2-associated binder protein 2 (Gab2) was shown to play an important role in G-CSF induced myeloid differentiation (Zhu et al. Blood 2004). Ligand stimulation of the G-CSF receptor results in the rapid phosphorylation of Gab2, but the identity of the responsible kinases and the molecular events dependent on Gab2 phosphorylation remain unclear. Because Janus kinases (Jaks) play a central role in G-CSF signaling, we investigated the involvement of Jaks in G-CSF-stimulated Gab2 phosphorylation using the hematologic DT40 cell line stably transduced with the human G-CSF receptor (DT40GR). Antisense Jak1 and Jak2 constructs expressed in DT40GR cells each produced a marked reduction in their target Jak protein, but only antisense Jak2 reduced G-CSF-stimulated Gab2 phosphorylation. To determine whether Gab2 phosphorylation required Jak2 kinase activity, dominant negative Jak2 mutants lacking catalytic activity were expressed in the DT40GR cells. Expression of dominant negative Jak2 inhibited Gab2 phosphorylation in response to G-CSF. Similarly, treatment with the Jak2-selective kinase inhibitor AG490 markedly reduced G-CSF-dependent Gab2 phosphorylation. Co-immunoprecipitation studies further demonstrated a G-CSF- and Gab2 phosphorylation-dependent association of Jak2 with Gab2 in vivo, which was detectable by 30 seconds after G-CSF stimulation. To determine whether Gab2 was a direct substrate of Jak2, we performed in vitro phosphorylation studies using Gab2-GST fusion protein substrates. Jak2 immunoprecipitated from G-CSF-stimulated cells, but not from control cells, phosphorylated the Gab2 fusion protein. To identify potential Jak2 tyrosine phosphorylation sites in Gab2, we used site-directed mutagenesis to produce three Gab2 tyrosine mutants. Tyrosines 409, 452, and 476 were each replaced by phenylalanine (Y409F, Y452F, and Y476F). The Y452F and Y476F mutations of Gab2 each inhibited G-CSF-stimulated Jak2-dependent phosphorylation of Gab2, both in stably-transfected DT40GR cells and in transiently-transfected 293 cells also transduced with the G-CSF receptor. In contrast, G-CSF-stimulated Gab2 phosphorylation appeared unaffected by the Y409F mutation. We also evaluated downstream events in G-CSF signaling in cells expressing these Gab2 tyrosine- mutants. Akt and Erk phosphorylation following G-CSF stimulation was inhibited by both the Y452F and Y476F Gab2 mutations, but was unaffected by the Y409F mutation. These results suggest that Jak2 may mediate G-CSF differentiation signals through Stat-independent mechanisms.

Reelin Dab-bles in the Brain

The secreted protein Reelin controls neuron positioning during embryonic brain development. The intracellular response to Reelin includes phosphorylation of the cytoplasmic protein Dab1 by Src-family tyrosine kinases. To further characterize the Reelin-Dab1 signaling pathway that controls neuronal migration, Ballif et al. identified proteins associated with phosphorylated Dab1 in the mouse brain during cortical development. Dab1-associated proteins were identified by mass spectrometry and included Crk-family proteins. Cultured cortical neurons treated with Reelin stimulated association of endogenous Crk proteins with phosphorylated Dab1. The interaction required phosphorylation of two of four potential tyrosine-phosphorylation sites in Dab1, and the authors speculate that Crk protein dimers may associate with the phosphorylated residues through an SH2 domain present on each monomer. The authors further determined that in response to Reelin, Crk proteins associate with phosphorylated C3G, an exchange protein that acts on the small GTP-binding protein Rap1. An increase in activated, GTP-bound Rap1 was also observed. Rap1 regulates cytoskeletal dynamics and migration and may thus may be a critical downstream effector of a Reelin-Dab1-Crk-C3G signaling pathway that controls neuron placement in the brain. B. A. Ballif, L. Arnaud, W. T. Arthur, D. Guris, A. Imamoto, J. A. Cooper, Activation of a Dab1/CrkL/C3G/Rap1 pathway in Reelin-stimulated neurons. Curr. Biol. 14 , 606-610 (2004). [Online Journal]

Stimulation of hTAF II68 (NTD)-mediated transactivation by v-Src

The three genes hTAF II68 , EWS, and TLS (called the TET family) encode related RNA binding proteins containing an RNA recognition motif and three glycine-, arginine-, and proline-rich regions in the C-terminus and a degenerated repeat containing the consensus sequence Ser-Tyr-Gly-Gln-Ser in the N-terminus. In many human cancers, the N-terminal portion of hTAF II68, EWS, or TLS is fused to the DNA binding domain of one of several transcription factors including Fli-1, ERG, ETV1, E1AF, WT1, ATF-1, CHOP, or TEC. We have recognized the presence of several potential tyrosine phosphorylation sites within the amino-terminal domain of hTAF II68 and have investigated the potential effects of cytoplasmic signaling on hTAF II68 function. Herein, we find that hTAF II68 is phosphorylated on tyrosine residue(s) by ectopic expression of v-Src protein tyrosine kinase in vitro and in vivo. The hTAF II68 protein can associated with the SH3 domains of several cell signaling proteins, including v-Src protein tyrosine kinase. We also document that full-length v-Src can stimulate hTAF II68-mediated transcriptional activation, whereas deletion mutants of v-Src are unable to exert this effect. In addition, cellular Src activity appears important for hTAF II68 function since hTAF II68-mediated transactivation is reduced in a dose-dependent fashion by ectopic overexpression of a dominant-negative mutant of Src. Taken together, our results suggest that the biological activities of hTAF II68 are linked to the cytoplasmic Src signal transduction pathway.

Motif analysis of the tumor suppressor gene MMAC/PTEN identifies tyrosines critical for tumor suppression and lipid phosphatase activity.

The tumor suppressor gene, MMAC/PTEN, has phosphatase, C2, and PDZ-binding domains as well as potential sites of regulation by phosphorylation, including tyrosine phosphorylation, which may contribute to its ability to modulate cell growth and viability. Several obvious and significant motifs were found in MMAC/PTEN, including most notably, a catalytic domain of tyrosine phosphatase (IHCxxGxxRS/T) and several potential tyrosine phosphorylation sites. To examine the functional significance of tyrosine phosphorylation of MMAC/PTEN, retroviral constructs were generated with mutations at two putative tyrosine phosphorylation sites (Y240A/Y240F and Y315A/Y315F). Stable expression of wild-type MMAC/PTEN in U251 human glioma cells (which do not normally produce a functional MMAC/PTEN gene product) resulted in a significant reduction of tumor growth in nude mice, decreased growth rate, saturation density, and colony formation in vitro, as well as dephosphorylation of D3-phosphorylated phosphatidylinositols (PtdIns) in vitro. Mutation of Y240 or Y315 to either alanine or phenylalanine abrogated the ability of MMAC/PTEN to alter growth rate, saturation density, and colony formation in vitro. The ability of MMAC/PTEN to limit tumor growth in nude mice was markedly decreased but not abrogated by mutation of Y240 or Y315 to alanine. Thus, Y240 and Y315 are required for MMAC/PTEN to decrease tumor growth in vitro and in vivo. In contrast to wild-type MMAC/PTEN, mutant MMAC/PTEN containing Y240A or Y315A was unable to dephosphorylate D3-phosphorylated PtdIns in vitro. Thus, Y240A and Y315A are involved in the ability of MMAC/PTEN to dephosphorylate PtdIns and regulate tumor cell growth in vitro and in vivo.