SH3-binding Motif Research Articles

Identification of an SH3-Binding Motif in a New Class of Methionine Aminopeptidases from Mycobacterium tuberculosis Suggests a Mode of Interaction with the Ribosome,

The crystal structure of the methionine aminopeptidase (MetAP) from Mycobacterium tuberculosis (MtMetAP1c) has been determined in the apo- and methionine-bound forms. This is the first structure of a type I MetAP with a significant extension at the amino terminus. The catalytic domain is similar to that of Escherichia coli MetAP (EcMetAP), and the additional 40-residue segment wraps around the surface with an extended but well-defined structure. There are several members of the actinomyces family of bacteria that contain MetAPs with such N-terminal extensions, and we classify these as MetAP type Ic (MetAP1c). Some members of this family of bacteria also contain a second MetAP (type Ia) similar in size to EcMetAP. The main difference between the apo- and the methionine-bound forms of MtMetAP1c is in the conformation of the metal-binding residues. The position of the methionine bound in the active site is very similar to that found in many of the known members of this family. Side chains of several residues in the S1 and S1' subsites shift as much as 1.5 A compared to EcMetAP. Residues 14-17 have the sequence Pro-Thr-Arg-Pro and adopt the conformation of a polyproline II helix. Model-building suggests that this PxxP segment can bind to an SH3 protein motif. Other type Ib and type Ic MetAPs with N-terminal extensions contain similarly located PxxP motifs. Also, several ribosomal proteins are known to include SH3 domains, one of which is located close to the tunnel from which the nascent polypeptide chain exits the ribosome. Therefore, it is proposed that the binding of MetAPs to the ribosome is mediated by a complex between a PxxP motif on the protein and an SH3 domain on the ribosome. It is also possible that zinc-finger domains, which are located at the extreme N-terminus of type I MetAPs, may participate in interactions with the ribosome.

Expression, purification and crystallization of a human protein SH3BGRL at atomic resolution.

The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 A. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.

Expression of TARSH gene in MEFs senescence and its potential implication in human lung cancer

To reveal the molecular mechanism of cellular senescence, we have surveyed the genes that are specifically upregulated via MEFs senescence by suppression subtractive hybridization method. We show here that mTARSH was induced particularly in the relative early phase of MEFs cellular senescence. Further structural analysis of mTARSH disclosed five splicing variants shared a common reading frame whose diversity was derived from the SH3-binding motif cluster in the middle of the gene. We also show that mTARSH mRNA predominantly expressed in lung and that conspicuous expression of TARSH was drastically declined in all several lung cancer cell lines we tested. Thus, TARSH presumably represents a trigger gene for evoking cellular senescence, which has also been suggested to be involved in the prevention of tumorigenesis.

The carboxy‐terminal tail region of human Cav2.1 (P/Q‐type) channel is not an essential determinant for its subcellular localization in cultured neurones

A recent report on the mechanism of synaptic targeting of Ca(v)2.2 channel suggested that this process depends upon the presence of long C-terminal tail and that protein interactions mediated by SH3-binding and PDZ-binding motifs in the tail region are important. To examine the possibility that C-terminal tail of the Ca(v)2.1 channel and the polyglutamine stretch therein are also involved in the mechanism for channel localization, we constructed several expression plasmids for human Ca(v)2.1 channel tagged with enhanced green fluorescent protein (EGFP) and introduced them into mouse hippocampal neuronal culture. HC construct encodes short version of Ca(v)2.1, and HS and HL encode Ca(v)2.1 channel with a long C-terminal tail, which contains polyglutamine tract of 13 (normal range) and 28 (SCA6 disease range) repeat units, respectively. Surprisingly, transfection with HC, HS, and HL gave essentially the same results: EGFP signal was observed in cell soma, dendrites, and the axon as well. Furthermore, mutation of the PDZ-binding motif located at the C-terminus of the long version of Ca(v)2.1, by adding FLAG tag, did not affect the localization patterns of HS and HL as well. Therefore, the C-terminal region is not indispensable for the subcellular localization of Ca(v)2.1 channel, nor expansion of polyglutamine length affected the localization of the channel. Thus, it is possible that the localization mechanism of Ca(v)2.1 channel is different from that of Ca(v)2.2, though these channels share various structural and functional characteristics.

Fully automated synthesis of (phospho)peptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization

BackgroundSynthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src.ResultsThe overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR) autophosphorylation site (pY1173) antibody ELISA (9H2) was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phospho)peptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of enzyme.ConclusionsThe ability of this method to interface with various robotics and instrumentation is highly flexible since the microtiter plate is an industry standard. It is highly scalable by increasing the surface area within the well or the number of wells and does not require specialized robotics. The microtiter plate array system is well suited to the study of protein kinase substrates, antigens, binding molecules, and inhibitors since these all can be quantitatively studied at a single uniform, reproducible interface.

Modulation of T-cell receptor signal transduction by herpesvirus signaling adaptor protein.

Because of its central regulatory role, T-cell receptor (TCR) signal transduction is a common target of viruses. We report here the identification of a small signaling protein, ORF5, of the T-lymphotropic tumor virus herpesvirus saimiri (HVS). ORF5 is predicted to contain 89 to 91 amino acids with an amino-terminal myristoylation site and six SH2 binding motifs, showing structural similarity to cellular LAT (linker for activation of T cells). Sequence analysis showed that, despite extensive sequence variation, the myristoylation site and SH2 binding motifs were completely conserved among 13 different ORF5 isolates. Upon TCR stimulation, ORF5 was efficiently tyrosine phosphorylated and subsequently interacted with cellular SH2-containing signaling proteins Lck, Fyn, SLP-76, and p85 through its tyrosine residues. ORF5 expression resulted in the marked augmentation of TCR signal transduction activity, evidenced by increased cellular tyrosine phosphorylation, intracellular calcium mobilization, CD69 surface expression, interleukin-2 production, and activation of the NF-AT, NF-kappa B, and AP-1 transcription factors. Despite its structural similarity to cellular LAT, however, ORF5 could only partially substitute for LAT function in TCR signal transduction. These results demonstrate that HVS utilizes a novel signaling protein, ORF5, to activate TCR signal transduction. This activation probably facilitates viral gene expression and, thereby, persistent infection.

Dok-1 tyrosine residues at 336 and 340 are essential for the negative regulation of Ras-Erk signalling, but dispensable for rasGAP-binding.

Dok-1 is a common substrate of many protein tyrosine kinases (PTKs). It recruits rasGAP and other SH2-containing proteins and negatively regulates Ras-Erk signalling downstream of PTKs. However, the mechanisms of its inhibitory effect are yet unclear. Here, a series of C-terminal deletion mutants of Dok-1 delineated the core domain for the inhibition of Erk from 334 to 346 amino acid, which contains two SH2-binding motifs having Tyr-336 or Tyr-340. The Dok-1 mutants having tyrosine-to-phenylalanine (YF) substitution(s) at Tyr-336 and/or Tyr-340 lost their inhibitory effect on Ras and Erk downstream of Src-like PTK, Lyn or Fyn, whereas the rasGAP-binding of each mutant remained intact. However, the Dok-1 mutant having YF substitutions at the rasGAP-binding sites (Tyr-295 and Tyr-361) also showed incapability of Ras and Erk inhibition. Moreover, the Dok-1 mutant having YF substitutions at Tyr-336 and Tyr-340 showed an impaired inhibitory effect on v-Abl-induced transformation of NIH-3T3 cells. These results demonstrate that Tyr-336 and Tyr-340 of Dok-1 are dispensable for rasGAP-binding but essential for inhibition of Ras-Erk signalling and cellular transformation downstream of PTKs. Thus, Dok-1 probably recruits as yet unidentified molecule(s), which, in concert with rasGAP, negatively regulate Ras-Erk signalling.

Activation of Stat3 transcription factor by Herpesvirus saimiri STP-A oncoprotein.

The saimiri transforming protein (STP) oncogene of Herpesvirus saimiri subgroup A strain 11 (STP-A11) is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. We previously showed that STP-A11 interacts with cellular Src kinase through its SH2 binding motif and that this interaction elicits Src signal transduction. Here we demonstrate that STP-A11 interacts with signal transducer and activator of transcription 3 (Stat3) independently of Src association and that the amino-terminal short proline-rich motif of STP-A11 and the central linker region of Stat3 are necessary for their interaction. STP-A11 formed a triple complex with Src kinase and Stat3 where Src kinase phosphorylated Stat3, resulting in the nuclear localization and transcriptional activation of Stat3. Consequently, the constitutively active Stat3 induced by STP-A11 elicited cellular signal transduction, which ultimately induced cell survival and proliferation upon serum deprivation. Furthermore, this activity was strongly correlated with the induction of Fos, cyclin D1, and Bcl-XL expression. These results demonstrate that STP-A11 independently targets two important cellular signaling molecules, Src and Stat3, and that these proteins cooperate efficiently to induce STP-A11-mediated transformation.

A novel crosstalk mechanism between nuclear receptor-mediated and growth factor/Ras-mediated pathways through PNRC-Grb2 interaction.

It has been demonstrated that proline-rich nuclear receptor coregulatory protein (PNRC) is a nuclear receptor coactivator that interacts with nuclear receptors through an SH3-binding motif located in its C-terminus. In the present report, a physical interaction between PNRC and Grb2 (an adapter protein involved in growth factor/Ras-mediated pathways) has been demonstrated using the GST pull-down assay, the yeast two-hybrid assay, as well as by coimmunoprecipitation. Cotransfection and fluorescence imaging have also confirmed the colocalization of PNRC and Grb2 in mammalian cells. Transient transfection experiments have demonstrated that, by interacting with each other, Grb2 decreases the coactivator activity of PNRC for nuclear receptors, and that PNRC suppresses Grb2-mediated Ras/MAP-kinase activation. Furthermore, it was discovered that HeLa cells overexpressing PNRC grew more slowly when compared to matched controls. Additionally, using a RT-PCR analysis of mRNA on six pairs of cancer/noncancer tissues, PNRC expression was found to be significantly lower in breast cancer tissue than in noncancer tissue. Based on these findings, we believe that PNRC and Grb2, by interacting with each other, can suppress nuclear receptor-mediated regulation and growth factor-mediated regulation in human breast tissue. This is a newly identified crosstalk mechanism for modulating these two important types of regulatory pathways.

Retrovirus-Induced Lung Cancer: Mechanisms of Transformation of Alveolar Type II Epithelial Cells

The jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, which is the only known example of a retrovirus-induced lung cancer. The finding that the JSRV env gene is sufficient to induce the transformation of murine and avian fibroblast cell lines has led to an investigation of the mechanism by which this gene induces transformation. Reports from this and other laboratories have presented conflicting data regarding the requirement for a known SH2-binding motif, which is found within the transmembrane domain of the JSRV env gene, in cellular transformation.

The Kaposi’s Sarcoma-Associated Herpesvirus (KSHV/HHV-8) K1 Protein Induces Expression of Angiogenic and Invasion Factors

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) has been linked to Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. In addition to endothelial cells and B lymphocytes, KSHV also has been shown to infect epithelial cells and keratinocytes. The transmembrane glycoprotein K1, encoded by the first open reading frame of KSHV, is a signaling protein capable of eliciting B-cell activation. We show that KSHV K1 can induce expression and secretion of vascular endothelial growth factor (VEGF) in epithelial and endothelial cells. Up-regulation of VEGF was mediated at the transcriptional level because expression of K1 resulted in VEGF promoter activation. We also show that K1 induces expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. Additional analyses with K1 mutant proteins revealed that the SH2 binding motifs present in the K1 cytoplasmic tail are necessary for VEGF secretion and MMP-9 induction. These results indicate that K1 signaling may contribute to KSHV-associated pathogenesis through a paracrine mechanism by promoting the secretion of VEGF and MMP-9 into the surrounding matrix.

ARHGAP8 is a novel member of the RHOGAP family related to ARHGAP1/CDC42GAP/p50RHOGAP: mutation and expression analyses in colorectal and breast cancers

The RHO family of small GTPases has multiple functions, including regulation of cytoskeletal organization, cell cycle progression and cell migration, among others. The key members of this family are RHO, RAC and CDC42. Active GTP-bound RHO proteins are down-regulated by RHO GTPase-activating proteins (RHOGAPs). Herein, we describe the identification, characterization and mutational analysis of a novel RHOGAP designated as ARHGAP8, which is located within a critical region of loss-of-heterozygosity on chromosome 22q13.31 in breast and colorectal carcinomas. ARHGAP8 shares an identical genomic organization with ARHGAP1/CDC42GAP/p50RHOGAP and the corresponding proteins share the same functional domains that distinguish them from other ARHGAP members. These domains include the C-terminal RHOGAP domain, a central SH3-binding motif, and an N-terminal BNIP-2/CDC42GAP homology (BCH)/Sec14p-like domain. Three alternatively spliced ARHGAP8 transcripts were expressed in normal mammary gland and colon, which differed in the size of the BCH/Sec14p-like domain only. PCR-SSCP analyses revealed a total of six germline missense variants in individuals with colorectal or breast cancer; however, somatic mutations were not identified. Surprisingly, ARHGAP8 expression was up-regulated in the majority of primary colorectal tumors analyzed. Taken together, ARHGAP8 encodes a novel RHOGAP with unique functional domains that is highly homologous to ARHGAP1/CDC42GAP/p50RHOGAP.

Specificity by Exclusion

If interaction of signaling proteins is determined through binding of interaction domains that potentially recognize domains in many other proteins, how is inappropriate cross talk of signaling pathways avoided? For example, the yeast osmosensor Sho has an SH3 domain that recognizes Pro-X-X-Pro motifs, and Sho's specific interaction partner Pbs2 has an SH3 binding motif. But yeast have 26 other known SH3 domains that occur in many proteins. One solution to the problem, now proposed by Zarrinpar et al. , is that negative selection actually removes potentially competing domains. The evidence consistent with such a strategy came from experiments in which the authors replaced the SH3 domain of Sho with each of the other 26 possible domains and looked for interaction of the modified protein with Sho's biological partner Pbs2. None of the other domains from yeast allowed detectable interaction in vitro or would support the osmolarity-sensing signaling pathway in vivo in which Sho and Pbs2 normally function. However, 6 out of 12 SH3 domains swiped from proteins belonging to other organisms did allow protein interaction in vitro and restoration of at least partial biological function in vivo. Expression of a Sho mutant that bound with high affinity to Pbs2, but also showed interaction with other SH3 domains, reduced fitness in yeast. Together the results suggest that, in this case, specificity has been generated by negative selection, which may have simply removed potential cross-reacting SH3-binding partners. Endy and Yaffe provide commentary. A. Zarrinpar, S.-H. Park, W. A. Lim, Optimization of specificity in a cellular protein interaction network by negative selection. Nature 426 , 676-680 (2003). [Online Journal] D. Endy, M. B. Yaffe, Molecular monogamy. Nature 426 , 614-615 (2003). [Online Journal]

Crystal structure of the tyrosine kinase domain of the hepatocyte growth factor receptor c-Met and its complex with the microbial alkaloid K-252a.

The protooncogene c-met codes for the hepatocyte growth factor receptor tyrosine kinase. Binding of its ligand, hepatocyte growth factor/scatter factor, stimulates receptor autophosphorylation, which leads to pleiotropic downstream signaling events in epithelial cells, including cell growth, motility, and invasion. These events are mediated by interaction of cytoplasmic effectors, generally through Src homology 2 (SH2) domains, with two phosphotyrosine-containing sequence motifs in the unique C-terminal tail of c-Met (supersite). There is a strong link between aberrant c-Met activity and oncogenesis, which makes this kinase an important cancer drug target. The furanosylated indolocarbazole K-252a belongs to a family of microbial alkaloids that also includes staurosporine. It was recently shown to be a potent inhibitor of c-Met. Here we report the crystal structures of an unphosphorylated c-Met kinase domain harboring a human cancer mutation and its complex with K-252a at 1.8-A resolution. The structure follows the well established architecture of protein kinases. It adopts a unique, inhibitory conformation of the activation loop, a catalytically noncompetent orientation of helix alphaC, and reveals the complete C-terminal docking site. The first SH2-binding motif (1349YVHV) adopts an extended conformation, whereas the second motif (1356YVNV), a binding site for Grb2-SH2, folds as a type II Beta-turn. The intermediate portion of the supersite (1353NATY) assumes a type I Beta-turn conformation as in an Shc-phosphotyrosine binding domain peptide complex. K-252a is bound in the adenosine pocket with an analogous binding mode to those observed in previously reported structures of protein kinases in complex with staurosporine.

Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism.

The selective RNA-binding protein QKI is essential for myelination in the central nervous system (CNS). QKI belongs to the family of signal transduction activators of RNA (STARs), characteristic of binding RNA and signaling molecules, therefore is postulated to regulate RNA homeostasis in response to developmental signals. Here we report that QKI acts downstream of the Src family protein tyrosine kinases (Src-PTKs) during CNS myelination. QKI selectively interacted with the mRNA encoding the myelin basic protein (MBP). Such interaction stabilized MBP mRNA and was required for the rapid accumulation of MBP mRNA during active myelinogenesis. We found that the interaction between QKI and MBP mRNA was negatively regulated by Src-PTK-dependent phosphorylation of QKI. During early myelin development, tyrosine phosphorylation of QKI in the developing myelin drastically declined, presumably leading to enhanced interactions between QKI and MBP mRNA, which was associated with the rapid accumulation of MBP mRNA and accelerated myelin production. Therefore, developmental regulation of Src-PTK-dependent tyrosine phosphorylation of QKI suggests a novel mechanism for accelerating CNS myelinogenesis via regulating mRNA metabolism.

Gads/Grb2-mediated association with LAT is critical for the inhibitory function of Gab2 in T cells.

A docking protein, Gab2, is recruited to the vicinity of the TCR complex and inhibits downstream signaling by interaction with negative regulators. However, the molecular mechanisms of this recruitment remain unclear. We have found that Gab2 associates with LAT upon TCR stimulation and that LAT is essential for Gab2 phosphorylation. By analysis of several Gab2 mutants, the c-Met binding domain (MBD) of Gab2 was found to be both necessary and sufficient for stimulation-induced LAT binding. Within the MBD domain, a novel Grb2 SH3 binding motif, PXXXR, is critical for constitutive association with Gads/Grb2. Through this association, Gab2 is recruited to the lipid raft after TCR ligation and exerts inhibitory function. The in vivo significance of this association is illustrated by the fact that T-cell responses are impaired in transgenic mice expressing wild-type Gab2 but not in mice expressing mutant Gab2 lacking the motif. Furthermore, T cells from Gab2-deficient mice showed enhanced proliferative responses upon TCR stimulation. These results indicate that Gads/Grb2-mediated LAT association is critical for the inhibitory function of Gab2, implying that Gab2 induced in stimulated T cells may exert an efficient negative feedback loop by recruiting inhibitory molecules to the lipid raft and competing with SLP-76 through Gads binding.

New Src-Family Kinase Activator

Members of the Src familiy of protein kinases contribute to signals emanating from various receptors on the cell membrane, but the mechanisms by which the receptors modulate kinase activity is unclear. Members of the Src family show interactions between protein domains that are thought to control kinase activity. In fact, the SH3 (Src homology 3) domain binds to a linker region within the protein and this binding favors an inactive conformation of the kinase. However, the sequence that is bound is actually not an optimal SH3-binding motif, so other higher affinity SH3-binding targets can potentially compete for binding and relieve this inhibition. In fact, activation of the Src family member Lck by the receptors CD28 or CD2 appears to be mediated by binding of an SH3 ligand in the receptor to the SH3 domain of the kinase. Now Cen et al. have discovered a new activator of Src-like kinases, Unc119, that appears to work in a similar manner. Unc119 interacts with the interleukin 5 (IL-5) receptor in a two hybrid screen and in vivo in human eosinophils. Unc119 contains both SH2- and SH3-binding motifs and interacts with the tyrosine kinase Lyn through these domains. Such binding of Unc119 to Lyn increased kinase activity or the enzyme. IL-5 signals through Lyn to promote survival of eosinophils. Eosinophils that took up Unc119 that had been modified to contain nuclear localization signals and a retroviral gp41 fusion domain showed increased activity of Lyn and prolonged survival. On the contrary, when cells took up antibody to Unc119 in similar experiments, Il-5-induced survival was reduced. Thus, Unc119 may represent a new class of regulatory protein for Src family kinases. O. Cen, M. M. Gorska, S. J. Stafford, S. Sur, R. Alam, Identification of Unc119 as a novel activator of SCR-type tyrosine kinases. J. Biol. Chem. 278 , 8837-8845 (2003). [Abstract] [Full Text]

Role of TFG sequences outside the coiled-coil domain in TRK-T3 oncogenic activation.

The TRK-T3 oncoprotein, isolated from a human papillary thyroid tumor, arises from the fusion between the N-terminal domain of the TFG gene and the tyrosine kinase domain of the NTRK1 receptor. The 68 kDa TRK-T3 oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain, which mediates protein oligomerization essential for the oncogene constitutive activation, and several consensus sites for protein interaction. In this study, we investigate the role of TFG sequences outside the coiled-coil domain on TRK-T3 activation, We constructed four mutants carrying different deletions of TFG sequences and expressed them in mammalian cells. By performing biochemical and biological assays we demonstrated that all the deleted regions are required for TRK-T3 activation, as they are involved in different mechanisms such as protein processing, formation of stable and/or functional complexes, and possible interaction with other proteins. By constructing site-specific mutants, we demonstrated a crucial role for a PB1 domain and a considerable contribution of an SH2-binding motif in TRK-T3 oncogenic activation. This work establishes an important role for TFG sequences outside the coiled-coil domain in the activation of the thyroid TRK-T3 oncogene.

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines, we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat mta1 gene, we cloned the human MTA1 gene and found it to be over-expressed in a variety of human cell lines (breast, ovarian, lung, gastric and colorectal cancer but not melanoma or sarcoma) and cancerous tissues (breast, esophageal, colorectal, gastric and pancreatic cancer). We found a close similarity between the human MTA1 and rat mta1 genes (88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively). Both genes encode novel proteins that contain a proline rich region (SH3-binding motif), a putative zinc finger motif, a leucine zipper motif and 5 copies of the SPXX motif found in gene regulatory proteins. Using Southern blot analysis the MTA1 gene was highly conserved, and using Northern blot analysis MTA1 transcripts were found in virtually all human cell lines (melanoma, breast, cervix and ovarian carcinoma cells and normal breast epithelial cells). However, the expression level of the MTA1 gene in normal breast epithelial cells was approximately 50% of that found in rapidly growing adenocarcinoma and atypical epithelial cell lines. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in inhibition of growth and invasion of human MDA-MB-231 breast cancer cells with relatively high MTA1 expression. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected with a mammalian expression vector containing a full-length MTA1 gene. Although some MTA1 protein was found in the cytoplasm, the vast majority of MTA1 protein was localized in the nucleus. Examination of recombinate MTA1 and related MTA2 proteins suggests that MTA1 protein is a histone deacetylase. It also appears to behave like a GATA-element transcription factor, since transfection of a GATA-element reporter into MTA1-expressing cells resulted in 10-20-fold increase in reporter expression over poorly MTA1-expressing cells. Since it was reported that nucleosome remodeling histone deacetylase complex (NuRD complex) involved in chromatin remodeling contains MTA1 protein and a MTA1-related protein (MTA2), we examined NuRD complexes for the presence of MTA1 protein and found an association of this protein with histone deacetylase. The results suggest that the MTA1 protein may serve multiple functions in cellular signaling, chromosome remodeling and transcription processes that are important in the progression, invasion and growth of metastatic epithelial cells.

Interaction of nebulin SH3 domain with titin PEVK and myopalladin: implications for the signaling and assembly role of titin and nebulin

Skeletal muscle nebulin is thought to determine thin filament length and regulate actomyosin interaction in a calcium/calmodulin or S100 sensitive manner. We have investigated the binding of nebulin SH3 with proline-rich peptides derived from the 28-mer PEVK modules of titin and the Z-line protein myopalladin, using fluorescence, circular dichroism and nuclear magnetic resonance techniques. Of the six peptides studied, PR2 of titin (VPEKKAPVAPPK) and myopalladin MyoP2 (646VKEPPPVLAKPK657) bind to nebulin SH3 with micromolar affinity (∼31 and 3.4 μM, respectively), whereas the other four peptides bind weakly (>100 μM). Sequence analysis of titins reveals numerous SH3 binding motifs that are highly enriched in the PEVK segments of titin isoforms. Our findings suggest that titin PEVK and myopalladin may play signaling roles in targeting and orientating nebulin to the Z-line during sarcomere assembly.