Functional role of cell surface CUB domain-containing protein 1 in tumour cell dissemination

The function of CUB domain-containing protein 1 (CDCP1), a recently described transmembrane protein expressed on the surface of hematopoietic stem cells and normal and malignant cells of different tissue origin, is not well defined. The contribution of CDCP1 to tumor metastasis was analyzed by using HeLa carcinoma cells overexpressing CDCP1 (HeLa-CDCP1) and a high-disseminating variant of prostate carcinoma PC-3 naturally expressing high levels of CDCP1 (PC3-hi/diss). CDCP1 expression rendered HeLa cells more aggressive in experimental metastasis in immunodeficient mice. Metastatic colonization by HeLa-CDCP1 was effectively inhibited with subtractive immunization-generated, CDCP1-specific monoclonal antibody (mAb) 41-2, suggesting that CDCP1 facilitates relatively late stages of the metastatic cascade. In the chick embryo model, time- and dose-dependent inhibition of HeLa-CDCP1 colonization by mAb 41-2 was analyzed quantitatively to determine when and where CDCP1 functions during metastasis. Quantitative PCR and immunohistochemical analyses indicated that CDCP1 facilitated tumor cell survival soon after vascular arrest. Live cell imaging showed that the function-blocking mechanism of mAb 41-2 involved enhancement of tumor cell apoptosis, confirmed by attenuation of mAb 41-2–mediated effects with the caspase inhibitor z-VAD-fmk. Under proapoptotic conditions in vitro, CDCP1 expression conferred HeLa-CDCP1 cells with resistance to doxorubicin-induced apoptosis, whereas ligation of CDCP1 with mAb 41-2 caused additional enhancement of the apoptotic response. The functional role of naturally expressed CDCP1 was shown by mAb 41-2–mediated inhibition of both experimental and spontaneous metastasis of PC3-hi/diss. These findings confirm that CDCP1 functions as an antiapoptotic molecule and indicate that during metastasis CDCP1 facilitates tumor cell survival likely during or soon after extravasation.

Cellular settings mediating Src Substrate switching between Focal Adhesion Kinase Tyrosine 861 and CUB-domain-containing protein 1 (CDCP1) Tyrosine 734

Reciprocal interactions between Src family kinases (SFKs) and focal adhesion kinase (FAK) are critical during changes in cell attachment. Recently it has been recognized that another SFK substrate, CUB-domain-containing protein 1 (CDCP1), is differentially phosphorylated during these events. However, the molecular processes underlying SFK-mediated phosphorylation of CDCP1 are poorly understood. Here we identify a novel mechanism in which FAK tyrosine 861 and CDCP1-Tyr-734 compete as SFK substrates and demonstrate cellular settings in which SFKs switch between these sites. Our results show that stable CDCP1 expression induces robust SFK-mediated phosphorylation of CDCP1-Tyr-734 with concomitant loss of p-FAK-Tyr-861 in adherent HeLa cells. SFK substrate switching in these cells is dependent on the level of expression of CDCP1 and is also dependent on CDCP1-Tyr-734 but is independent of CDCP1-Tyr-743 and -Tyr-762. In HeLa CDCP1 cells, engagement of SFKs with CDCP1 is accompanied by an increase in phosphorylation of Src-Tyr-416 and a change in cell morphology to a fibroblastic appearance dependent on CDCP1-Tyr-734. SFK switching between FAK-Tyr-861 and CDCP1-Tyr-734 also occurs during changes in adhesion of colorectal cancer cell lines endogenously expressing these two proteins. Consistently, increased p-FAK-Tyr-861 levels and a more epithelial morphology are seen in colon cancer SW480 cells silenced for CDCP1. Unlike protein kinase Cδ, FAK does not appear to form a trimeric complex with Src and CDCP1. These data demonstrate novel aspects of the dynamics of SFK-mediated cell signaling that may be relevant during cancer progression.

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a Tm of 64.9 degrees C and a deltaHm of 259.7 kcal mol(-1). The linear dependence of deltaHm on Tm was used to estimate a value of deltaCp of 7.9 kcal mol(-1) K(-1) or 21.3 cal (mol of residue)(-1) K(-1). These values are higher than the corresponding deltaCp's for most globular proteins studied to date. However, the extrapolated values of deltaH and deltaS (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the deltaCp (per mol of residue) and that this temperature increases with an increase in deltaCp. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

Characterization of single-stranded DNA-binding proteins from Mycobacteria. The carboxyl-terminal of domain of SSB is essential for stable association with its cognate RecA protein

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Limiting the Extent of the RDN1 Heterochromatin Domain by a Silencing Barrier and Sir2 Protein Levels in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, transcriptional silencing occurs at the cryptic mating-type loci (HML and HMR), telomeres, and ribosomal DNA ( rDNA; RDN1). Silencing in the rDNA is unusual in that polymerase II (Pol II) promoters within RDN1 are repressed by Sir2 but not Sir3 or Sir4. rDNA silencing unidirectionally spreads leftward, but the mechanism of limiting its spreading is unclear. We searched for silencing barriers flanking the left end of RDN1 by using an established assay for detecting barriers to HMR silencing. Unexpectedly, the unique sequence immediately adjacent to RDN1, which overlaps a prominent cohesin binding site (CARL2), did not have appreciable barrier activity. Instead, a fragment located 2.4 kb to the left, containing a tRNA(Gln) gene and the Ty1 long terminal repeat, had robust barrier activity. The barrier activity was dependent on Pol III transcription of tRNA(Gln), the cohesin protein Smc1, and the SAS1 and Gcn5 histone acetyltransferases. The location of the barrier correlates with the detectable limit of rDNA silencing when SIR2 is overexpressed, where it blocks the spreading of rDNA heterochromatin. We propose a model in which normal Sir2 activity results in termination of silencing near the physical rDNA boundary, while tRNA(Gln) blocks silencing from spreading too far when nucleolar Sir2 pools become elevated.

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules,naiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular studies on LIM-domain protein CRP1

Various intrinsic and external factors are constantly attacking the cells causing damage to DNA and to other cellular structures. Cells in turn have evolved with different kinds of mechanisms to protect against the attacks and to repair the damage. Ultraviolet radiation (UVR) is one of the major environmental genotoxic carcinogens that causes inflammation, mutations, immunosuppression, accelerated aging of the skin and skin cancers. Epidermis is the outermost layer of the skin consisting mostly of keratinocytes, whose primary function is to protect the skin against e.g. UV radiation. LIM domain proteins are a group of proteins involved in regulation of cell growth, damage signalling, cell fate determination and signal transduction. Despite their two zinc fingers, LIM domains do not bind to DNA, but rather mediate protein-protein interactions and function as modular protein binding interfaces. We initially identified CSRP1 as UVR-regulated transcript by using expression profiling. Here we have further studied the regulation and function of CRP1, a representative of cysteine rich protein- family consisting of two LIM domains. We find that CRP1 is increased by UVR in primary human keratinocytes and in normal human skin fibroblasts. Ectopic expression of CRP1 protected the cells against UVR and provided a survival advantage, whereas silencing of CRP1 rendered the cells more photosensitive. Actinic keratosis is a premalignant lesion of skin caused by excess exposure to sunlight and sunburn, which may lead to formation of squamous cell carcinoma. The expression of CRP1 was increased in basal keratinocytes of Actinic keratosis patient specimens suggesting that CRP1 may be increased by constant exposure to UVR and may provide survival advantage for the cells also in vivo. In squamous cell carcinoma, CRP1 was only expressed in the fibroblasts surrounding the tumour. Moreover, we found that ectopic expression of CRP1 suppresses cell proliferation. Transforming growth factor beta (TGFbeta) is a multifunctional cytokine that regulates several functions in cell including growth, apoptosis and differentiation, and plays important roles in pathological disorders like cancer and fibrosis. We found that TGFbeta-signalling pathway regulates CRP1 at protein, but not at transcriptional level. The increase was mediated both through Smad and non-Smad signalling pathways involving MAPK/p38. Furthermore, we found that TGFbeta-mediated increase in CRP1 was associated with myofibroblast differentiation, and that CRP1 was significantly more expressed in idiopathic pulmonary fibrosis as compared to normal lung specimens. Since cell contractility is a distinct feature of myofibroblasts, and CRP1 is associated with actin cytoskeleton, we studied the role of CRP1 in cell contractility. CRP1 was found to localize to stress fibres that mediate contractility and to mediate myofibroblast contraction. These studies identify CRP1 as a stress responsive and cytokine regulated cytoskeletal protein that participates in pathological processes involved in fibrotic diseases and cancer.

Characterization of the DNA-binding domain of beta protein, a component of phage lambda Red-pathway by UV catalyzed cross-linking

beta protein, a key component of Red-pathway of phage lambda is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli. To facilitate studies on structure-function relationships, we overexpressed beta protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of beta protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of beta protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that beta protein formed stable nucleoprotein complexes with 36-mer, but not with 27- or 17-mer DNA. Interestingly, the interaction of beta protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgCl2, and the binding was abolished by 250 mM NaCl. The K-d of beta protein binding to 36-mer DNA was on the order of 1.8 x 10(-6) M. Photochemical cross-linking of native beta protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding oi the structure and function of beta protein.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

Modulation of Catalytic Activity in Multi-Domain Protein Tyrosine Phosphatases

Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.