p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: A third member of the p53 family?

p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a sequence-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5′-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T)(T/A)GPyPyPy-3′. We have now identified, partially purified, and characterized an additional ≈40-kDa nuclear protein, p53CP (p53 competing protein), that specifically binds to the consensus p53 binding sites found in several p53 downstream target genes, including Waf-1, Gadd45, Mdm2, Bax, and RGC. The minimal sequence requirement for binding is a 14-bp motif, 5′-CTTGCTTGAACAGG-3′ [5′-C(A/T)(T/A)GPyPyPyPuPuPuC(A/T)(T/A)G-3′], which includes the central nucleotides of the typical p53 binding site with one mismatch. p53CP and p53 (complexed with antibody) showed a similar binding specificity to Waf-1 site but differences in Gadd45 and T3SF binding. Like p53, p53CP also binds both double- and single-stranded DNA oligonucleotides. Important to note, cell cycle blockers and DNA damaging reagents, which induce p53 binding activity, were found to inhibit p53CP binding in p53-positive, but not in p53-negative, cells. This finding suggested a p53-dependent coordinate regulation of p53 and p53CP in response to external stimuli. p53CP therefore could be a third member of the p53 family, in addition to p53 and p73, a newly identified p53 homolog. p53CP, if sequestering p53 from its DNA binding sites through competitive binding, may provide a novel mechanism of p53 inactivation. Alternatively, p53CP may have p53-like functions by binding and transactivating p53 downstream target genes. Cloning of the p53CP gene ultimately will resolve this issue.

The Rat Myosin myr 5 Is a GTPase-activating Protein for Rho In Vivo: Essential Role of Arginine 1695

myr 5 is an unconventional myosin (class IX) from rat that contains a Rho-family GTPase-activating protein (GAP) domain. Herein we addressed the specificity of the myr 5 GAP activity, the molecular mechanism by which GAPs activate GTP hydrolysis, the consequences of myr 5 overexpression in living cells, and its subcellular localization. The myr 5 GAP activity exhibits a high specificity for Rho. To achieve similar rates of GTPase activation for RhoA, Cdc42Hs, and Rac1, a 100-fold or 1000-fold higher concentration of recombinant myr 5 GAP domain was needed for Cdc42Hs or Rac1, respectively, as compared with RhoA. Cell lysates from Sf9 insect cells infected with recombinant baculovirus encoding myr 5 exhibited increased GAP activity for RhoA but not for Cdc42Hs or Rac1. Analysis of Rho-family GAP domain sequences for conserved arginine residues that might contribute to accelerate GTP hydrolysis revealed a single conserved arginine residue. Mutation of the corresponding arginine residue in the myr 5 GAP domain to a methionine (M1695) virtually abolished Rho-GAP activity. Expression of myr 5 in Sf9 insect cells induced the formation of numerous long thin processes containing occasional varicosities. Such morphological changes were dependent on the myr 5 Rho-GAP activity, because they were induced by expressing the myr 5 tail or just the myr 5 Rho-GAP domain but not by expressing the myr 5 myosin domain. Expression of myr 5 in mammalian normal rat kidney (NRK) or HtTA-1 HeLa cells induced a loss of actin stress fibers and focal contacts with concomitant morphological changes and rounding up of the cells. Similar morphological changes were observed in HtTA-1 HeLa cells expressing just the myr 5 Rho-GAP domain but not in cells expressing myr 5 M1695. These morphological changes induced by myr 5 were inhibited by coexpression of RhoV14, which is defective in GTP hydrolysis, but not by RhoI117. myr 5 was localized in dynamic regions of the cell periphery, in the perinuclear region in the Golgi area, along stress fibers, and in the cytosol. These results demonstrate that myr 5 has in vitro and in vivo Rho-GAP activity. No evidence for a Rho effector function of the myr 5 myosin domain was obtained.

A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins

The Krüppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys2-His2 type. Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region. KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys3HisCys4, family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction. The KRIP-1 protein has high homology to TIF1, a putative modulator of ligand-dependent activation function of nuclear receptors. A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied. When a GAL4–KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription. Thus, KRIP-1 interacts with the KRAB-A region of C2H2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

An electric lobe suppressor for a yeast choline transport mutation belongs to a new family of transporter-like proteins

Choline is an important metabolite in all cells due to the major contribution of phosphatidylcholine to the production of membranes, but it takes on an added role in cholinergic neurons where it participates in the synthesis of the neurotransmitter acetylcholine. We have cloned a suppressor for a yeast choline transport mutation from a Torpedo electric lobe yeast expression library by functional complementation. The full-length clone encodes a protein with 10 putative transmembrane domains, two of which contain transporter-like motifs, and whose expression increased high-affinity choline uptake in mutant yeast. The gene was called CTL1 for its choline transporter-like properties. The homologous rat gene, rCTL1, was isolated and found to be highly expressed as a 3.5-kb transcript in the spinal cord and brain and as a 5-kb transcript in the colon. In situ hybridization showed strong expression of rCTL1 in motor neurons and oligodendrocytes and to a lesser extent in various neuronal populations throughout the rat brain. High levels of rCTL1 were also identified in the mucosal cell layer of the colon. Although the sequence of the CTL1 gene shows clear homology with a single gene in Caenorhabditis elegans, several homologous genes are found in mammals (CTL2–4). These results establish a new family of genes for transporter-like proteins in eukaryotes and suggest that one of its members, CTL1, is involved in supplying choline to certain cell types, including a specific subset of cholinergic neurons.

The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein α subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein β subunit (Gβ5L). RGS9 and Gβ5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein γ-subunit like domain that can mediate their association with Gβ5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307–13312). We report an example of such a complex whose cellular localization and function are clearly defined.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.

Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor.

The APC variants I1307K and E1317Q are associated with colorectal tumors, but not always with a family history

Classical familial adenomatous polyposis (FAP) is a high-penetrance autosomal dominant disease that predisposes to hundreds or thousands of colorectal adenomas and carcinoma and that results from truncating mutations in the APC gene. A variant of FAP is attenuated adenomatous polyposis coli, which results from germ-line mutations in the 5′ and 3′ regions of the APC gene. Attenuated adenomatous polyposis coli patients have “multiple” colorectal adenomas (typically fewer than 100) without the florid phenotype of classical FAP. Another group of patients with multiple adenomas has no mutations in the APC gene, and their phenotype probably results from variation at a locus, or loci, elsewhere in the genome. Recently, however, a missense variant of APC (I1307K) was described that confers an increased risk of colorectal tumors, including multiple adenomas, in Ashkenazim. We have studied a set of 164 patients with multiple colorectal adenomas and/or carcinoma and analyzed codons 1263–1377 (exon 15G) of the APC gene for germ-line variants. Three patients with the I1307K allele were detected, each of Ashkenazi descent. Four patients had a germ-line E1317Q missense variant of APC that was not present in controls; one of these individuals had an unusually large number of metaplastic polyps of the colorectum. There is increasing evidence that there exist germ-line variants of the APC gene that predispose to the development of multiple colorectal adenomas and carcinoma, but without the florid phenotype of classical FAP, and possibly with importance for colorectal cancer risk in the general population.

Phosphatidylinositol 3-kinase-γ activates Bruton’s tyrosine kinase in concert with Src family kinases

Bruton’s tyrosine kinase (Btk) is essential for normal B lymphocyte development and function. The activity of Btk is partially regulated by transphosphorylation within its kinase domain by Src family kinases at residue Tyr-551 and subsequent autophosphorylation at Tyr-223. Activation correlates with Btk association with cellular membranes. Based on specific loss of function mutations, the Btk pleckstrin homology (PH) domain plays an essential role in this activation process. The Btk PH domain can bind in vitro to several lipid end products of the phosphatidylinositol 3-kinase (PI 3-kinase) family including phosphatidylinositol 3,4,5-trisphosphate. Activation of Btk as monitored by elevation of phosphotyrosine content and a cellular transformation response was dramatically enhanced by coexpressing a weakly activated allele of Src (E378G) and the two subunits of PI 3-kinase-γ. This activation correlates with new sites of phosphorylation on Btk identified by two-dimensional phosphopeptide mapping. Activation of Btk was dependent on the catalytic activity of all three enzymes and an intact Btk PH domain and Src transphosphorylation site. These combined data define Btk as a downstream target of PI 3-kinase-γ and Src family kinases.

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP4) from InsP5. Additionally, mIPMK forms InsP4 from Ins(1,4,5)P3 and InsP5 from Ins(1,3,4,5)P4.

Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain

Cyclin-dependent kinase (Cdk) 5 is a unique member of the Cdk family, because Cdk5 kinase activity is detected only in the nervous tissue. Two neuron-specific activating subunits of Cdk5, p35 and p39, have been identified. Overlapping expression pattern of these isoforms in the embryonic mouse brain and the significant residual Cdk5 kinase activity in brain homogenate of the p35−/− mice indicate the redundant functions of the Cdk5 activators in vivo. Severe neuronal migration defects in p35−/−Cdk5 +/− mice further support the idea that the redundant expression of the Cdk5 activators may cause a milder phenotype in p35−/− mice compared with Cdk5−/− mice. Mutant mice lacking either Cdk5 or p35 exhibit certain similarities with Reelin/Dab1-mutant mice in the disorganization of cortical laminar structure in the brain. To elucidate the relationship between Cdk5/p35 and Reelin/Dab1 signaling, we generated mouse lines that have combined defects of these genes. The addition of heterozygosity of either Dab1 or Reelin mutation to p35−/− causes the extensive migration defects of cortical neurons in the cerebellum. In the double-null mice of p35 and either Dab1 or Reelin, additional migration defects occur in the Purkinje cells in the cerebellum and in the pyramidal neurons in the hippocampus. These additional defects in neuronal migration in mice lacking both Cdk5/p35 and Reelin/Dab1 indicate that Cdk5/p35 may contribute synergistically to the positioning of the cortical neurons in the developing mouse brain.

Integrin-mediated Adhesion Regulates Cell Polarity and Membrane Protrusion through the Rho Family of GTPasesV⃞

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through α5β1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma

Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10−/− mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10−/− mice suggests that there is a considerable redundancy in the keratin gene family.

A unique human gene that spans over 230 kb in the human chromosome 8p11-12 and codes multiple family proteins sharing RNA-binding motifs.

A unique gene, RBP-MS, spanning over 230 kb in the human chromosome 8p11-12 near the Werner syndrome gene locus is described. The single-copy RBP-MS gene is alternatively spliced, resulting in a family of at least 12 transcripts (average length of 1.5 kb). Nine different types of cDNAs that encode an RNa-binding motif at the N terminus and helix-rich sequences at the C terminus have been identified thus far. Among the 16 exons identified, four 5'-proximal exons contained sequences homologous to the RNA-binding domain of Drosophila couch potato gene. Northern blot analysis showed that the RBP-MS gene was expressed strongly in the heart, prostate, intestine, and ovary, and poorly in the skeletal muscle, spleen, thymus, brain, and peripheral leukocytes. The possible role of this gene in RNA metabolism is discussed.