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At least 253 records · Page 14Linked to original sources
PubMed2003

What can developmental disorders tell us about the neurocomputational constraints that shape development? The case of Williams syndrome.

The uneven cognitive phenotype in the adult outcome of Williams syndrome has led some researchers to make strong claims about the modularity of the brain and the purported genetically determined, innate specification of cognitive modules. Such arguments have particularly been marshaled with respect to language. We challenge this direct generalization from adult phenotypic outcomes to genetic specification and consider instead how genetic disorders provide clues to the constraints on plasticity that shape the outcome of development. We specifically examine behavioral studies, brain imaging, and computational modeling of language in Williams syndrome but contend that our theoretical arguments apply equally to other cognitive domains and other developmental disorders. While acknowledging that selective deficits in normal adult patients might justify claims about cognitive modularity, we question whether similar, seemingly selective deficits found in genetic disorders can be used to argue that such cognitive modules are prespecified in infant brains. Cognitive modules are, in our view, the outcome of development, not its starting point. We note that most work on genetic disorders ignores one vital factor, the actual process of ontogenetic development, and argue that it is vital to view genetic disorders as proceeding under different neurocomputational constraints, not as demonstrations of static modularity.

Cognition Disorders↗
PubMed1995

Loss of heterozygosity of imprinted genes in SV40 t/T antigen-induced hepatocellular carcinomas.

Expression of the chromosomally linked Insulin-like Growth Factor II (IGF-II) and H19 genes is regulated by parental imprinting during development, since the maternally inherited IGF-II and the paternally inherited H19 alleles are inactive in fetal tissues. Here we show that expression of IGF-II and H19 genes is activated in transgenic mice during SV40 Tag-induced hepatocarcinogenesis and that imprinting of both genes is conserved in the liver tumors. Allelic imbalances of IGF-II and H19 genes and other chromosome 7 markers were detected in one third (13/39) of the hepatocellular carcinomas analysed. A strong bias on the allele retained in the neoplasms was observed, since underrepresentation or complete loss of maternal chromosome 7 was recognised in 12/13 cases. High levels of IGF-II mRNA were expressed by all carcinomas with relative excess of paternal chromosome 7 alleles and suppressed H19 expression was found in the neoplasms lacking the maternal alleles. Overall the results indicate that expression of imprinted genes is involved in progression of experimental liver tumors and suggest that the murine chromosome 7, whose loss may possibly cause the inactivation of a growth-inhibitory gene, is preferentially retained as paternal copy in the liver tumors because of parental imprinting of IGF-II gene.

Alleles↗
PubMed2006

The Drosophila p21 activated kinase Mbt regulates the actin cytoskeleton and adherens junctions to control photoreceptor cell morphogenesis.

The p21 activated kinase (Pak) family of protein kinases are involved in many cellular functions like re-organisation of the cytoskeleton, transcriptional control, cell division, and survival. These pleiotropic actions are reflected in a plethora of known interacting proteins and phosphorylation substrates. Yet, the integration of a single Pak protein into signalling pathways controlling a particular developmental process are less well studied. For two of the three known Pak proteins in Drosophila melanogaster, D-Pak and Mbt, distinct functions during eye development have been established. In this study we undertook a genetic approach to identify proteins acting in the Mbt signalling pathway during photoreceptor cell morphogenesis. The genetic screen identified the actin depolymerisation factor Twinstar/Cofilin as one target of Mbt signalling. Twinstar/Cofilin becomes phosphorylated upon activation of Mbt. However, biochemical and genetic experiments question the role of the LIM domain protein kinase (Limk) as a major link between Mbt and Twinstar/Cofilin as it has been suggested for other PAK proteins. Constitutive activation of Mbt not only disturbs the actin cytoskeleton but also affects adherens junction organisation indicating a requirement of the protein in cell adhesion dependent processes during photoreceptor cell differentiation.

Actins↗
PubMed2000

Paxillin interactions.

UNLABELLED: Cell Science at a Glance on the Web: electronic copies of the full-size poster insert are available in various formats Paxillin is a multi-domain protein that localizes in cultured cells primarily to sites of cell adhesion to the extracellular matrix (ECM) called focal adhesions. Focal adhesions form a structural link between the ECM and the actin cytoskeleton and are also important sites of signal transduction; their components propagate signals arising from the activation of integrins following their engagement with ECM proteins, such as fibronectin, collagen and laminin. Importantly, focal adhesion proteins including paxillin also serve as a point of convergence for signals resulting from stimulation of various classes of growth factor receptor. Paxillin localizes to focal adhesions through its LIM domains, possibly through a direct association with &bgr;-integrin tails or an intermediate protein 'X'. In lymphoid cells it can bind directly to *(4)-integrin (not shown). Its primary function is as a molecular adapter or scaffold protein that provides multiple docking sites at the plasma membrane for an array of signaling and structural proteins. For example, it provides a platform for protein tyrosine kinases such as FAK and SRC, which are activated as a result of adhesion or growth factor stimulation. Phosphorylation of residues in the N-terminus of paxillin by these kinases permits the regulated recruitment of downstream effector molecules such as CRK, which (via association with CAS) is important for transduction of external signals into changes in cell motility and for modulation of gene expression by the various MAP kinase cascades. LIM-domain-associated kinases regulate recruitment of paxillin to focal adhesions. In addition, negative regulators of these pathways, including CSK (an inhibitor of SRC activity) and PTP-PEST (a phosphatase that dephosphorylates p130Cas), bind directly to paxillin, thereby bringing them into close proximity with their targets. Paxillin binds to many proteins that are involved in effecting changes in the organization of the actin cytoskeleton, which are necessary for cell motility events associated with embryonic development, wound repair and tumor metastasis. These range from structural proteins such as vinculin and actopaxin that bind actin directly to regulators of actin cytoskeletal dynamics such as the ARF GAP, PKL, the exchange factor PIX and the p21-activated kinase, PAK. These proteins serve as modulators/effectors of the ARF and RHO GTPase families. Several of the paxillin-binding proteins have oncogenic equivalents, such as v-Src, v-Crk and BCR-ABL (not shown). These proteins probably use paxillin both as a substrate and as a docking site to perturb, and even bypass, the normal adhesion and growth factor signaling cascades necessary for controlled cell proliferation. Others, such as the E6 protein from Papillomavirus, facilitate transformation by disrupting the normal links between paxillin and the actin cytoskeleton by displacing paxillin-LD-motif-binding proteins. ABBREVIATIONS: CAS, CRK-associated substrate; CH, calponin-homology domain; CSK, C-terminal SRC kinase; E6, Papillomavirus E6 protein; FAK, focal adhesion kinase; GIT, GRK interacter; GPCR, heterotrimeric-G-protein-coupled receptor; GRK, G-protein-coupled-receptor kinase; MAPK, mitogen-activated protein kinase (ERK, p38, JNK); PAK, p21-activated kinase; PBS, paxillin-binding subdomain; PIX, PAK-interacting exchange factor; PKL, paxillin kinase linker; POR1, partner of Rac; PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine; RTK, growth factor receptor tyrosine kinase; SH, SRC-homology domain.

Cells, Cultured↗
PubMed2004

Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes.

The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling.

Animals↗
PubMed1995

Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro.

We have isolated the murine Limk1 gene, which is a single copy gene located at the distal end of mouse chromosome 5. Limk1 exhibits a 95% homology to the human homologue, LIMK, which contains two LIM domains and a putative protein kinase domain. Although Limk1 and LIMK contain all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro and that mutation of residue D460 within the IHRDL motif abolishes kinase activity. Northern blot showed preferential expression of a 3.5 kb message in adult spinal cord and brain. In situ hybridisation confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerve and dorsal root ganglia. Limk1 also contains two tandem LIM-domains. These zinc-finger like domains can mediate protein-protein interactions and have been described in nuclear and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signalling can be integrated.

Amino Acid Sequence↗
PubMed2002

Clik1: a novel kinase targeted to actin stress fibers by the CLP-36 PDZ-LIM protein.

In this report we have characterized a novel, ubiquitously expressed kinase, Clik1, that is predominantly nuclear and undergoes autophosphorylation. Yeast two-hybrid analysis indicated a highly specific association between Clik1 and CLP-36, which was identified in 36 out of 37 Clik1-interacting clones. CLP-36 is a PDZ-LIM protein that localizes to actin stress fibers in nonmuscle cells and associates with alpha-actinin via its PDZ-domain. The association of CLP-36 with Clik1, in turn, is mediated by the C-terminal part of CLP-36 containing the LIM domain, and association was not noted with the closely related ALP PDZ-LIM protein. Interestingly, the association with CLP-36 led to relocalization of the otherwise nuclear Clik1 kinase to actin stress fibers, where it disrupted the periodic staining pattern of CLP-36. Taken together these results establish the CLP-36 PDZ-LIM protein as an adapter, recruiting the Clik1 kinase to actin stress fibers in nonmuscle cells, and suggest that Clik1 represents a novel regulator of actin stress fibers.

Actins↗
PubMed1999

Ajuba, a novel LIM protein, interacts with Grb2, augments mitogen-activated protein kinase activity in fibroblasts, and promotes meiotic maturation of Xenopus oocytes in a Grb2- and Ras-dependent manner.

LIM domain-containing proteins contribute to cell fate determination, the regulation of cell proliferation and differentiation, and remodeling of the cell cytoskeleton. These proteins can be found in the cell nucleus, cytoplasm, or both. Whether and how cytoplasmic LIM proteins contribute to the cellular response to extracellular stimuli is an area of active investigation. We have identified and characterized a new LIM protein, Ajuba. Although predominantly a cytosolic protein, in contrast to other like proteins, it did not localize to sites of cellular adhesion to extracellular matrix or interact with the actin cytoskeleton. Removal of the pre-LIM domain of Ajuba, including a putative nuclear export signal, led to an accumulation of the LIM domains in the cell nucleus. The pre-LIM domain contains two putative proline-rich SH3 recognition motifs. Ajuba specifically associated with Grb2 in vitro and in vivo. The interaction between these proteins was mediated by either SH3 domain of Grb2 and the N-terminal proline-rich pre-LIM domain of Ajuba. In fibroblasts expressing Ajuba mitogen-activated protein kinase activity persisted despite serum starvation and upon serum stimulation generated levels fivefold higher than that seen in control cells. Finally, when Ajuba was expressed in fully developed Xenopus oocytes, it promoted meiotic maturation in a Grb2- and Ras-dependent manner.

3T3 Cells↗
PubMed2005

The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex.

The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-kappaB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCzeta. Ajuba recruits TRAF6 to p62 and in vitro activates PKCzeta activity and is a substrate of PKCzeta. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-kappaB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-kappaB activity following IL-1 stimulation. We propose that Ajuba is a new cytosolic component of the IL-1 signaling pathway modulating IL-1-induced NF-kappaB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Trichostatin A-induced detransformation correlates with decreased focal adhesion kinase phosphorylation at tyrosine 861 in ras-transformed fibroblasts.

To elucidate the role of focal adhesion kinase (pp125FAK) in transformation, its phosphorylation in transformed fibroblasts was compared with that of detransformed fibroblasts induced by a histone deacetylase inhibitor, trichostatin A (TSA). Inhibition of histone deacetylase activity in two different ras-transformed fibroblast lines by TSA induced a morphological change into a flattened and more spread morphology, implying detransformation. These morphological changes included increased spreading ability of transformed NIH 3T3 cells on fibronectin. Of the six tyrosine phosphorylation sites in pp125FAK, phosphorylation at position 861 (Tyr-861) was clearly decreased during detransformation by TSA. It resulted from decreased activity of Src family tyrosine kinase and/or decreased amount of Src kinase interacting with pp125FAK. Furthermore, phosphorylation of Tyr-861 was reduced substantially by the Src family kinase inhibitor, PP1, while overexpression of Src kinase increased its phosphorylation, implying that Src kinase regulates phosphorylation of pp125FAK at Tyr-861. All of these findings suggest that increased phosphorylation of pp125FAK at Tyr-861 correlates with Ras-induced transformation of fibroblasts, and TSA is able to detransform them through regulation of pp125FAK phosphorylation at Tyr-861 by an Src family kinase.

3T3 Cells↗
PubMed2006

Phosphorylation at the hydrophobic site of protein kinase C Apl II is increased during intermediate term facilitation.

In Aplysia, persistent increases in synaptic strength are paralleled by the persistent activation of the novel protein kinase C Apl II. We raised a phosphospecific antibody against serine 725, the hydrophobic motif in protein kinase C Apl II. Phosphorylation of serine 725 increased in parallel to the persistent activation of the kinase. We expressed protein kinase C where this site was mutated to an alanine to prevent phosphorylation. The mutated protein kinase C showed decreased specific activity consistent with a model where the kinase is less stable in the absence of phosphorylation of this site. Endogenous phosphorylation of protein kinase C Apl II at serine 725 was unaffected by either activation of protein kinase C by phorbol esters, or inhibition of protein kinase C using two distinct inhibitors, suggesting the site is not autophosphorylated. Consistent with this, overexpressed kinase-dead protein kinase C Apl II still was phosphorylated at serine 725, although to a lesser extent than wild-type protein kinase C Apl II. While PDK appears to interact with the serine 725 site, it is not responsible for its phosphorylation. Finally inhibition of phosphoinositide-3 kinase or the target of rapamycin by pharmacological agents did not block basal phosphorylation of serine 725 in Aplysia ganglia. Our results suggest trans-phosphorylation of protein kinase C Apl II as Ser 725 occurs during persistent activation of the kinase, but this does not appear to be downstream of phosphoinositide-3 kinase.

Alkaloids↗
PubMed2000

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain.

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.

Amino Acid Sequence↗
PubMed1998

Nck-2, a novel Src homology2/3-containing adaptor protein that interacts with the LIM-only protein PINCH and components of growth factor receptor kinase-signaling pathways.

Many of the protein-protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-beta, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-beta was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain-containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways.

3T3 Cells↗
PubMed1999

The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells.

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. We report here that PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. Finally, we have found that PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and have detected clusters of PINCH that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Regulation of the neuronal actin cytoskeleton by ADF/cofilin.

Actin and microtubules are major cytoskeletal elements of most cells including neurons. In order for a cell to move and change shape, its cytoskeleton must undergo rearrangements that involve breaking down and reforming filaments. Many recent reviews have focused on the signaling pathways emanating from receptors that ultimately affect axon growth and growth cone steering. This particular review will address changes in the actin cytoskeleton modulated by the family of actin dynamizing proteins known as actin depolymerizing factor (ADF)/cofilin or AC proteins. Though much is known about inactivation of AC proteins through phosphorylation at ser3 by LIM or TES kinases, new mechanisms of regulation of AC have recently emerged. A novel phosphatase, slingshot (SSH), and the 14-3-3 family of regulatory proteins have also been found to affect AC activity. The potential role of AC proteins in modulating the actin organizational changes that accompany neurite initiation, axonogenesis, growth cone guidance, and dendritic spine formation will be discussed.

Actin Depolymerizing Factors↗
PubMed2003

Identification of genes in thyrocytes regulated by unfolded protein response by using disulfide bond reducing agent of dithiothreitol.

Disulfide bonds are formed between the sulfhydryl groups in two cysteine residues of a protein. The formation of these bonds is necessary for the proper folding of a protein into its active three-dimensional form. In this study, the genes associated with disulfide bond formation of proteins from the rat thyroid cell line, FRTL-5 cell, were investigated using disulfide bond reducing agent of dithiothreitol (DTT), which prevented disulfide formation of newly synthesized proteins. The expression of six genes, they being the cAMP phosphodiesterase 7A1, neuronal cell death inducible putative kinase (NIPK), cytosolic LIM protein (Ajuba), Eker, early growth response 1 and the ferritin heavy chain, was specifically enhanced under both reductive conditions and various endoplasmic reticulum (ER) stresses inducing drugs such as Brefeldin A (BFA), calcium ionophore A23187 (A23187) and tunicamycin. These results suggest that a suitable redox environment is necessary for the correct disulfide bond conformation in thyrocytes in a complex system.

Animals↗