Family Of Adaptor Proteins Research Articles

Functional Mechanisms and Roles of Adaptor Proteins in Abl-Regulated Cytoskeletal Actin Dynamics

Abl is a nonreceptor tyrosine kinase and plays an essential role in the modeling and remodeling of F-actin by transducing extracellular signals. Abl and its paralog, Arg, are unique among the tyrosine kinase family in that they contain an unusual extended C-terminal half consisting of multiple functional domains. This structural characteristic may underlie the role of Abl as a mediator of upstream signals to downstream signaling machineries involved in actin dynamics. Indeed, a group of SH3-containing accessory proteins, or adaptor proteins, have been identified that bind to a proline-rich domain of the C-terminal portion of Abl and modulate its kinase activity, substrate recognition, and intracellular localization. Moreover, the existence of signaling cascade and biological outcomes unique to each adaptor protein has been demonstrated. In this paper, we summarize functional roles and mechanisms of adaptor proteins in Abl-regulated actin dynamics, mainly focusing on a family of adaptor proteins, Abi. The mechanism of Abl's activation and downstream signaling mediated by Abi is described in comparison with those by another adaptor protein, Crk.

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

Models of Crk Adaptor Proteins in Cancer

The Crk family of adaptor proteins (CrkI, CrkII, and CrkL), originally discovered as the oncogene fusion product, v-Crk, of the CT10 chicken retrovirus, lacks catalytic activity but engages with multiple signaling pathways through their SH2 and SH3 domains. Crk proteins link upstream tyrosine kinase and integrin-dependent signals to downstream effectors, acting as adaptors in diverse signaling pathways and cellular processes. Crk proteins are now recognized to play a role in the malignancy of many human cancers, stimulating renewed interest in their mechanism of action in cancer progression. The contribution of Crk signaling to malignancy has been predominantly studied in fibroblasts and in hematopoietic models and more recently in epithelial models. A mechanistic understanding of Crk proteins in cancer progression in vivo is still poorly understood in part due to the highly pleiotropic nature of Crk signaling. Recent advances in the structural organization of Crk domains, new roles in kinase regulation, and increased knowledge of the mechanisms and frequency of Crk overexpression in human cancers have provided an incentive for further study in in vivo models. An understanding of the mechanisms through which Crk proteins act as oncogenic drivers could have important implications in therapeutic targeting.

Neuronal dCbl downregulates the EGFR‐ERK pathway and controls the production of Insulin‐like Peptides in Drosophila

The Cbl proteins are a family of E3 ubiquitin ligases and adaptor proteins that regulates a diversity of cellular signaling and play essential roles in growth, development and survival. To explore the mechanism by which Cbl exerts its regulatory actions in vivo, here we show that in the fruit fly Drosophila melanogaster, dCbl controls multiple aspects of physiological processes through downregulating the expression of insulin‐like peptides (dilps) in the brain. We found knockdown by RNA interference of the expression of dCbl in neurons or specifically in insulin‐producing cells led to increased sensitivity to oxidative or nutrient stresses, decreased carbohydrate storage, and shortened lifespan. Moreover, neuronal dCbl deficiency caused elevations in the p‐dAkt and p‐dERK, which was accompanied by increased expression of dilps. Interestingly, genetic interaction analysis showed blocking neuronal dEGFR‐dERK signaling abolished dCbl deficiency‐mediated upregulation of dilps. In parallel, suppression of c‐Cbl in rat insulin‐producing INS‐1 cells also increased the production of insulin in an ERK‐dependent manner. Collectively, these results reveal that Cbl downregulation of the EGFR pathway is coupled to the dynamic control of insulin biosynthesis, suggesting Cbl may act through an evolutionarily conserved mechanism to participate in systemic modulation of growth, metabolism and lifespan.

REEPs, an ER adapter protein family, have differential effects on GPCR expression

IntroductionREEPs are members of a growing class of proteins termed ER morphogens or adapter proteins. REEP1 mutations have been implicated in Hereditary Spastic Paraplegia (HSP). To further understand the role of REEPs in ER membrane trafficking and HSP, we have examined their role using two model proteins, alpha2A and alpha2C adrenergic receptors (ARs).MethodsTransiently transfected HEK293 cells expressing individual Flag‐REEP proteins and HA‐alpha2A&C ARs were examined by FACS analysis to determine REEP effects on receptor expression, correlated with ligand binding and deglycosylation analysis.ResultsAnalysis of individual cells expressing both REEPs and ARs revealed a complex effect. Co‐expression of alpha2A ARs with REEP1, REEP2, and REEP6 caused a small increase in plasma membrane AR expression (1.0×, 1.0×, and 0.5× respectively), however there was a larger increase in total AR expression within the cell (1.8×, 1.8×, and 1.2× respectively). A similar analysis of alpha2C AR expression showed a larger REEP effect on surface expression (1.3×, 1.4×, and 1.9× respectively) coupled with a smaller increase in total AR expression (1.2×, 1.2×, and 1.5× respectively). Ligand binding of total membranes showed a similar pattern. Lastly, deglycosylation analysis of ARs revealed that co‐expression of REEPs led to an increase in a minimally glycosylated form of alpha2C ARs.ConclusionsREEPs were originally identified by their ability to enhance GPCR surface expression; however, their effects cannot be generalized. REEPs appear to enhance the production or stability of a minimally glycosylated alpha2C AR. Lastly, co‐IP analysis has revealed that REEPs interact preferentially with alpha2C, but not alpha2A ARs.

Resveratrol or 32°C hypothermia applied during reperfusion after cardiac ischemia reduces mitochondrial translocation of p66shc

Reactive oxygen species (ROS) are important in ischemia (I) and reperfusion (RP) injury. Mitochondria are sources of ROS via electron leak from complexes I and III. P66shc, a splice variant of ShcA adaptor protein family, enhances mROS by oxidizing reduced cyt c and reducing O2 to H2O2. Mild hypothermia or resveratrol (RES) reduce ROS during IRP. Although p66shc ablation protected against cardiac IR injury, it is unknown if and when p66shc is activated during IRP, and if inhibiting p66shc activation on RP protects against IRP injury. To test this, we isolated and perfused guinea pig hearts with KR buffer for 55 min (time control, TC), or 20 min or 35 min ischemia, or 35 min ischemia plus 20 min RP, with or without hypothermia or RES (10 μM). We isolated heart tissue total cell lysates, mitochondria, and cytosol and measured levels of p66shc by Western blot (WB) with anti‐SHC antibody and phosphorylation of Serine36 of p66shc by immunoprecipitation with anti‐SHC followed by WB with p66‐ Ser36 antibody. We found that after RP mitochondrial p66shc increased two fold above TC while phosphorylation of Ser36 increased at 20 min ischemia, but decreased at 35 min ischemia, and increased again at 20 min RP in total cell lysates. Our results demonstrate that p66shc is activated by IRP, that activated p66shc translocates from cytosol to mitochondria, and that RES or hypothermia applied only during RP reduces mitochondrial p66shc to TC levels.

Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform

ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin.

Modeling and Simulation of Aggregation of Membrane Protein LAT with Molecular Variability in the Number of Binding Sites for Cytosolic Grb2-SOS1-Grb2

The linker for activation of T cells (LAT), the linker for activation of B cells (LAB), and the linker for activation of X cells (LAX) form a family of transmembrane adaptor proteins widely expressed in lymphocytes. These scaffolding proteins have multiple binding motifs that, when phosphorylated, bind the SH2 domain of the cytosolic adaptor Grb2. Thus, the valence of LAT, LAB and LAX for Grb2 is variable, depending on the strength of receptor activation that initiates phosphorylation. During signaling, the LAT population will exhibit a time-varying distribution of Grb2 valences from zero to three. In the cytosol, Grb2 forms 1∶1 and 2∶1 complexes with the guanine nucleotide exchange factor SOS1. The 2∶1 complex can bridge two LAT molecules when each Grb2, through their SH2 domains, binds to a phosphorylated site on a separate LAT. In T cells and mast cells, after receptor engagement, receptor phosphoyrlation is rapidly followed by LAT phosphorylation and aggregation. In mast cells, aggregates containing more than one hundred LAT molecules have been detected. Previously we considered a homogeneous population of trivalent LAT molecules and showed that for a range of Grb2, SOS1 and LAT concentrations, an equilibrium theory for LAT aggregation predicts the formation of a gel-like phase comprising a very large aggregate (superaggregate). We now extend this theory to investigate the effects of a distribution of Grb2 valence in the LAT population on the formation of LAT aggregates and superaggregate and use stochastic simulations to calculate the fraction of the total LAT population in the superaggregate.

CD2AP Regulates SUMOylation of CIN85 in Podocytes

Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.

MNT inhibits the migration of human hepatocellular carcinoma SMMC7721 cells

Max binding protein (MNT) is a member of the Myc/Max/Mad network that plays a role in cell proliferation, differentiation and apoptosis. We previously observed that MNT was differentially expressed in hepatocellular carcinoma (HCC) and interacted with Nck1 by 2-DE. Nck family adaptor proteins function to couple tyrosine phosphorylation signals, regulate actin cytoskeletal reorganization and lead to cell motility. In order to investigate the regulatory role of MNT in HCC migration, we used transient transfection with a MNT expressing vector to overexpress MNT protein in SMMC7721 cells, and MNT siRNA to knockdown MNT expression. Rho Family Small GTPase activation assay, Western blots and transwell assay were used to determine the migration potential of cells. We found that knockdown of MNT expression might promote SMMC7721 cell migration, while the overexpressed MNT could significantly inhibit cell migration. It further emphasized the role of MNT in inhibition of cell migration that might be a promising target for HCC chemotherapy.

The Gads Adaptor Protein Is Required to Mediate Lymphoid Disease Downstream of BCR-ABL,

Abstract 3750Chronic Myeloid Leukemia is mediated by the BCR-ABL chromosomal translocation. Animal modeling experiments utilizing retroviral transduction and subsequent bone marrow transplantation have demonstrated that BCR-ABL generates a robust myeloproliferative disease that is fatal in recipient animals within 21–28 days that have received transduced marrow from donor mice primed with 5-fluorouracil. Structure/function experiments revealed that mice receiving bone marrow expressing BCR-ABL Y177F generate a T cell lymphoma with a latency of 73–116 days, demonstrating that the SH2 proteins that bind to BCR-ABL pY177 are critical for promoting myeloproliferative disease.There are three members of the Grb2 adaptor protein family: Grb2, Gads and Grap. Biochemical experiments suggest that the Grb2 adaptor protein binds to BCR-ABL Y177 and acts to couple BCR-ABL to the Gab2 adaptor protein. In turn, Gab2 links to activation of the Ras and PI 3 kinase signal transduction pathways. Previous studies from our group revealed that Gads binds to BCR-ABL in vitro. We hypothesized that Gads plays an important role downstream of BCR-ABL. To test this objective, we performed retroviral transduction-bone marrow transplantation experiments utilizing bone marrow from wild type and Gads-deficient mice.Thymocytes from Gads−/− mice exhibit defective proliferation in response to CD3 activation and deficits in positive and negative selection. The loss of Gads results uncoupling of Slp-76 and Lat, and impaired PLCγ phosphorylation in thymocytes. No other prominent phenotypes have been documented in this animal model.We show that retroviral transduction-bone marrow transplantation experiments completed with 5-fluoruracil primed bone marrow display a similar latency in BCR-ABL expressing bone marrow from wild type or Gads-deficient mice.Retroviral transduction-bone marrow transplantation experiments can also be completed with normal (unprimed) bone marrow which generates a mixed syndrome comprising myeloproliferative disease, B cell Acute Lymphoid Leukemia and macrophage tumours with a latency from 30–70 days in mice transduced with BCR-ABL. Strikingly, we observe that the lymphoid disease is absent in experiments completed with Gads−/− bone marrow expressing BCR-ABL and all mice succumb to myeloproliferative disease at 21 days. These data suggest that Gads is required for lymphoproliferative disease in this model.We have examined signaling events in the CML-T1 and LAMA-84 CML cell lines that co-express GRB2 and GADS in order to develop models to account for our observations. GRB2 and GADS both co-immunoprecipitate BCR-ABL in both cell lines. However, there is remarkable specificity in the complexes that bind to each adaptor protein. For example, GRB2 co-immunoprecipitates the SHC adaptor protein (but not GAB2), whereas GADS co-immunoprecipitates GAB2 and SLP-76 from both CML cell lines. We are currently performing experiments to confirm these observations in BCR-ABL expressing Acute Lymphoid Leukemia patient specimens.Our data suggests that BCR-ABL mediates Y177-dependent signaling via Grb2-dependent activation of Ras and PI 3 kinase in a myeloproliferative model of CML. However, in keeping with the important role that Gads plays in maintaining lymphocyte homeostasis, Gads mediates lymphoproliferative disease downstream of BCR-ABL, through the recruitment of specific signaling intermediates. Disclosures:No relevant conflicts of interest to declare.

The SCF-Fbxo40 Complex Induces IRS1 Ubiquitination in Skeletal Muscle, Limiting IGF1 Signaling

Insulin-like growth factor 1 (IGF1) induces skeletal muscle hypertrophy by activating the IGF1R/IRS1/PI3K/Akt pathway. However the effect of IGF1 in differentiated muscle is limited by IRS1 ubiquitination and proteasome-mediated breakdown. In skeletal muscle, IGF1R activation sensitizes IRS1 to degradation, and a screen for the responsible E3 ligase identified Fbxo40 as mediating this rapid turnover of IRS1, since IRS1 loss can be rescued by knockdown of Fbxo40. In biochemical assays, an SCF E3 ligase complex containing Fbxo40 directly ubiquitinates IRS1, and this activity is enhanced by increased tyrosine phosphorylation of IRS1. Fbxo40 is muscle specific in expression and is upregulated during differentiation. Knockdown of Fbxo40 induces dramatic hypertrophy of myofibers. Mice null for Fbxo40 have increased levels of IRS1 and demonstrate enhanced body and muscle size during the growth phase associated with elevated IGF1 levels. These findings establish an important means of restraining IGF1's effects on skeletal muscle.

Wager of war on bromodomains

Dawson and colleagues showed that mixed-lineage leukaemia (MLL) fusion proteins, which commonly occur in aggressive leukaemia, associate with the bromodomain and extraterminal (BET) family of chromatin adaptor proteins. So, to target MLL-fusion-induced transcriptional responses, the authors treated mouse and human MLL-fusion leukaemia cells with a BET inhibitor, GSK1210151A, which induced cell cycle arrest and apoptosis. GSK1210151A treatment altered the expression of a common set of genes; in particular, BCL2, MYC and cyclin-dependent kinase 6 (CDK6) expression was reduced. The drug also improved the survival of mice with leukaemia induced by the expression of MLL-AF9 or MLL-AF4. In another paper, Mertz and colleagues treated various leukaemia and lymphoma cell lines with the BET inhibitor JQ1, which suppressed MYC expression and altered the expression of MYC target genes. Treatment with JQ1 resulted in cell cycle arrest and apoptosis, which was suppressed by exogenous expression of MYC from a BET regulation-resistant promoter. Finally, JQ1 reduced tumour growth of Burkitt's lymphoma cell and acute myeloid leukaemia cell xenografts. Together, these and the other recent papers that have investigated targeting the BET family indicate that this could be an efficacious strategy for targeting several types of leukaemia and, potentially, tumours overexpressing MYC.

The Adaptor Protein SH2B3 (Lnk) Negatively Regulates Neurite Outgrowth of PC12 Cells and Cortical Neurons

SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.

Regulation of G Protein-Coupled Receptor Function by Na+/H+ Exchange Regulatory Factors

Many G protein-coupled receptors (GPCR) exert patterns of cell-specific signaling and function. Mounting evidence now supports the view that cytoplasmic adapter proteins contribute critically to this behavior. Adapter proteins recognize highly conserved motifs such as those for Src homology 3 (SH3), phosphotyrosine-binding (PTB), and postsynaptic density 95/discs-large/zona occludens (PDZ) docking sequences in candidate GPCRs. Here we review the behavior of the Na+/H+ exchange regulatory factor (NHERF) family of PDZ adapter proteins on GPCR signalling, trafficking, and function. Structural determinants of NHERF proteins that allow them to recognize targeted GPCRs are considered. NHERF1 and NHERF2 are capable also of modifying the assembled complex of accessory proteins such as β-arrestins, which have been implicated in regulating GPCR signaling. In addition, NHERF1 and NHERF2 modulate GPCR signaling by altering the G protein to which the receptor binds or affect other regulatory proteins that affect GTPase activity, protein kinase A, phospholipase C, or modify downstream signaling events. Small molecules targeting the site of NHERF1-GPCR interaction are being developed and may become important and selective drug candidates.

Hierarchical Binding of Cofactors to the AAA ATPase p97

The hexameric AAA ATPase p97 is involved in several human proteinopathies and mediates ubiquitin-dependent protein degradation among other essential cellular processes. Via its N-terminal domain (N domain), p97 interacts with multiple regulatory cofactors including the UFD1/NPL4 heterodimer and members of the "ubiquitin regulatory X" (UBX) domain protein family; however, the principles governing cofactor selectivity remain to be deciphered. Our crystal structure of the FAS-associated factor 1 (FAF1)UBX domain in complex with the p97N domain reveals that the signature Phe-Pro-Arg motif known to be crucial for interactions of UBX domains with p97 adopts a cis-proline configuration, in contrast to a cis-trans mixture we derive for the isolated FAF1UBX domain. Biochemical studies confirm that binding critically depends on a proline at this position. Furthermore, we observe that the UBX proteins FAF1 and UBXD7 only bind to p97-UFD1/NPL4, but not free p97, thus demonstrating for the first time a hierarchy in p97-cofactor interactions.

Resistin Promotes Cardiac Hypertrophy via the AMP-activated Protein Kinase/Mammalian Target of Rapamycin (AMPK/mTOR) and c-Jun N-terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Coordinating electrical activity of the heart: ankyrin polypeptides in human cardiac disease

Introduction: Over the past ten years, ankyrin polypeptides have emerged as players in cardiac excitation–contraction coupling. Once thought to solely play a structural role, loss-of-function variants of genes encoding ankyrin polypeptides have highlighted how this protein mediates subcellular localization of various electrical components of the excitation–contraction coupling machinery. Evidence has revealed how disruption of this localization is the primary cause of various cardiomyopathies, ranging from long-QT syndrome 4, to sinus node disease, to more common forms of arrhythmias.Areas covered: The roles of ankyrin polypeptides in excitation–contraction coupling in the heart and the development of ankyrin-specific cardiomyopathies. How ankyrin polypeptides may be involved in structural and electrical remodeling of the heart, post-myocardial infarct. How ankyrin interactions with membrane-bound ion channels may regulate these channels' response to stimuli. New data, which offers the potential for unique therapies, for not only combating heart disease, but also for wider applications to various disease states.Expert opinion: The ankyrin family of adapter proteins is emerging as an intimate player in cardiac excitation–contraction coupling. Until recently, these proteins have gone largely unappreciated for their importance in proper cardiac function. New insights into how these proteins function within the heart are offering potentially new avenues for therapies against cardiomyopathy.

Identification of Dermcidin as a novel binding protein of Nck1 and characterization of its role in promoting cell migration

A distinct feature of hepatocellular carcinoma (HCC) is the tendency of tumor cells to disperse throughout the liver. Nck family adaptor proteins function to couple tyrosine phosphorylation signals to regulate actin cytoskeletal reorganization that leads to cell motility. In order to explore the role of Nck in HCC development, we performed GST pull-down assay using the SH2 domain of Nck1 as bait. The resulting precipitates were separated by 2-DE. Mass spectrometry analysis revealed a group of Nck1 SH2 domain-binding proteins that were differentially expressed in HCC. One of these proteins, dermcidin (DCD), and its interaction with Nck1, was further validated in vitro. GST pull-down assay revealed that Nck1 SH2 domain binds to the phosphotyrosine residue at position 20 (Y20) of the DCD. Pervandate treatment significantly enhanced the interaction between DCD and Nck1. Moreover, we demonstrated that forced expression of DCD could activate Rac1 and Cdc42 and promoted cell migration. Taken together, these data suggest a role of DCD in tumor metastasis.

Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells: Implications for Phenotype Modification

Kindlins are a novel family of intracellular adaptor proteins in integrin-containing focal adhesions. Kindlin-1 and -2 are expressed in the skin, but whether and how they cooperate in adult epithelial cells have remained elusive. We uncovered the overlapping roles of kindlin-1 and -2 in maintaining epithelial integrity and show that the phenotype of kindlin-1-deficient cells can be modulated by regulating kindlin-2 gene expression and vice versa. The experimental evidence is provided by use of human keratinocyte cell lines that express both kindlins, just kindlin-1 or kindlin-2, or none of them. Double deficiency of kindlin-1 and -2 had significant negative effects on focal adhesion formation and actin cytoskeleton organization, cell adhesion, survival, directional migration, and activation of β(1) integrin, whereas deficiency of one kindlin only showed variable perturbation of these functions. Cell motility and formation of cell-cell contacts were particularly affected by lack of kindlin-2. These results predict that kindlin-1 and -2 can functionally compensate for each other, at least in part. The high physiologic and pathologic significance of the compensation was emphasized by the discovery of environmental regulation of kindlin-2 expression. UV-B irradiation induced loss of kindlin-2 in keratinocytes. This first example of environmental regulation of kindlin expression has implications for phenotype modulation in Kindler syndrome, a skin disorder caused by kindlin-1 deficiency.