Oligomerization of activated d- and l-guanosine mononucleotides on templates containing d- and l-deoxycytidylate residues

The oligomerization of activated d- and l- and racemic guanosine-5′-phosphoro-2-methylimidazole on short templates containing d- and l-deoxycytidylate has been studied. Results obtained with d-oligo(dC)s as templates are similar to those previously reported for experiments with a poly(C) template. When one l-dC or two consecutive l-dCs are introduced into a d-template, regiospecific synthesis of 3′-5′ oligo(G)s proceeds to the end of the template, but three consecutive l-dCs block synthesis. Alternating d-,l-oligomers do not facilitate oligomerization of the d-, l-, and racemic 2-guanosine-5′-phosphoro-2-methylimidazole. We suggest that once a “predominately d-metabolism” existed, occasional l-residues in a template would not have led to the termination of self-replication.

The amphiphysin-like protein 1 (ALP1) interacts functionally with the cABL tyrosine kinase and may play a role in cytoskeletal regulation

cABL is a protooncogene, activated in a subset of human leukemias, whose protein product is a nonreceptor tyrosine kinase of unknown function. cABL has a complex structure that includes several domains and motifs found in proteins implicated in signal transduction pathways. An approach to elucidate cABL function is to identify proteins that interact directly with cABL and that may serve as regulators or effectors of its activity. To this end, a protein-interaction screen of a phage expression library was undertaken to identify proteins that interact with specific domains of cABL. An SH3-domain-containing protein has been identified that interacts with sequences in the cABL carboxyl terminus. The cDNA encoding ALP1 (amphiphysin-like protein 1) was isolated from a 16-day mouse embryo. ALP1 has high homology to BIN1, a recently cloned myc-interacting protein, and also shows significant homology to amphiphysin, a neuronal protein cloned from human and chicken. The amino terminus has homology to two yeast proteins, Rvs167 and Rvs161, which are involved in cell entry into stationary phase and cytoskeletal organization. ALP1 binds cABL in vitro and in vivo. Expression of ALP1 results in morphological transformation of NIH 3T3 fibroblasts in a cABL-dependent manner. The properties of ALP1 suggest that it may be involved in possible cytoskeletal functions of the cABL kinase. Additionally, these results provide further evidence for the importance of the cABL carboxyl terminus and its binding proteins in the regulation of cABL function.

Chemical complementation identifies a proton acceptor for redox-active tyrosine D in photosystem II

Through the use of site-directed mutagenesis and chemical rescue, we have identified the proton acceptor for redox-active tyrosine D in photosystem II (PSII). Effects of chemical rescue on the tyrosyl radical were monitored by EPR spectroscopy. We also have acquired the Fourier–transform infrared (FT-IR) spectrum associated with the oxidation of tyrosine D and concomitant protonation of the acceptor. Mutant and isotopically labeled PSII samples are used to assign vibrational lines in the 3,600–3,100 cm−1 region to N-H modes of His-189 in the D2 polypeptide. When His-189 in D2 is changed to a leucine (HL189D2) in PSII, dramatic alterations of both EPR and FT-IR spectra are observed. When imidazole is introduced into HL189D2 samples, results from both EPR and FT-IR spectroscopy argue that imidazole is functionally reconstituted into an accessible pocket and that imidazole acts as a chemical mimic for His-189. Small perturbations of EPR and FT-IR spectra are consistent with access to this pocket in wild-type PSII, as well. Structures of the analogous site in bacterial reaction centers suggest that an accessible pocket, large enough to contain imidazole, is bordered by tyrosine D and His-189 in the D2 polypeptide. These data provide evidence that His-189 in the D2 polypeptide of PSII acts as a proton acceptor for redox-active tyrosine D and that proton transfer to the imidazole ring facilitates the efficient oxidation/reduction of tyrosine D.

A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation

Sik, the mouse homologue of the breast tumor kinase Brk, is expressed in differentiating cells of the gastrointestinal tract and skin. We examined expression and activity of Sik in primary mouse keratinocytes and a mouse embryonic keratinocyte cell line (EMK). Calcium-induced differentiation of these cells has been shown to be accompanied by the activation of tyrosine kinases and rapid phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65). We demonstrate that Sik is activated within 2 min after calcium addition in primary keratinocytes and EMK cells. In EMK cells, Sik binds GAP-A.p65, and this interaction is mediated by the Sik Src homology 2 domain. Although Sik directly complexes with GAP-A.p65, overexpression of wild-type or kinase defective Sik in EMK cells does not lead to detectable changes in GAP-A.p65 phosphorylation. These data suggest that Sik is not responsible for phosphorylation of GAP-A.p65. GAP-A.p65 may act as an adapter protein, bringing Sik into proximity of an unidentified substrate. Overexpression of Sik in EMK cells results in increased expression of filaggrin during differentiation, supporting a role for Sik in differentiation.

Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1–4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1–5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1–3F) and FGFR1-Y463/583/585/766F mutant (FGFR1–4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1–3F, and FGFR1–4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Correlation of α-Fetoprotein Expression in Normal Hepatocytes during Development with Tyrosine Phosphorylation and Insulin Receptor Expression

The molecular mechanism of hepatic cell growth and differentiation is ill defined. In the present study, we examined the putative role of tyrosine phosphorylation in normal rat liver development and in an in vitro model, the α-fetoprotein-producing (AFP+) and AFP-nonproducing (AFP−) clones of the McA-RH 7777 rat hepatoma. We demonstrated in vivo and in vitro that the AFP+ phenotype is clearly associated with enhanced tyrosine phosphorylation, as assessed by immunoblotting and flow cytometry. Moreover, immunoprecipitation of proteins with anti-phosphotyrosine antibody showed that normal fetal hepatocytes expressed the same phosphorylation pattern as stable AFP+ clones and likewise for adult hepatocytes and AFP− clones. The tyrosine phosphorylation of several proteins, including the β-subunit of the insulin receptor, insulin receptor substrate-1, p85 regulatory subunit of phosphatidylinositol-3-kinase, and ras-guanosine triphosphatase-activating protein, was observed in AFP+ clones, whereas the same proteins were not phosphorylated in AFP− clones. We also observed that fetal hepatocytes and the AFP+ clones express 4 times more of the insulin receptor β-subunit compared with adult hepatocytes and AFP− clones and, accordingly, that these AFP+ clones were more responsive to exogenous insulin in terms of protein tyrosine phosphorylation. Finally, growth rate in cells of AFP+ clones was higher than that measured in cells of AFP− clones, and inhibition of phosphatidylinositol-3-kinase by LY294002 and Wortmannin blocked insulin- and serum-stimulated DNA synthesis only in cells of AFP+ clones. These studies provide evidences in support of the hypothesis that signaling via insulin prevents hepatocyte differentiation by promoting fetal hepatocyte growth.

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC–EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations ≥1 μg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 μg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, γ-catenin, and p120Cas. These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.

The Interaction of Activated Integrin Lymphocyte Function-associated Antigen 1 with Ligand Intercellular Adhesion Molecule 1 Induces Activation and Redistribution of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 in T Lymphocytes

Integrin receptors play a central role in the biology of lymphocytes, mediating crucial functional aspects of these cells, including adhesion, activation, polarization, migration, and signaling. Here we report that induction of activation of the β2-integrin lymphocyte function-associated antigen 1 (LFA-1) in T lymphocytes with divalent cations, phorbol esters, or stimulatory antibodies is followed by a dramatic polarization, resulting in a characteristic elongated morphology of the cells and the arrest of migrating lymphoblasts. This cellular polarization was prevented by treatment of cells with the specific tyrosine kinase inhibitor genistein. Furthermore, the interaction of the activated integrin LFA-1 with its ligand intercellular adhesion molecule 1 induced the activation of the cytoplasmic tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK-2). FAK activation reached a maximum after 45 min of stimulation; in contrast, PYK-2 activation peaked at 30 min, declining after 60 min. Upon polarization of lymphoblasts, FAK and PYK-2 redistributed from a diffuse localization in the cytoplasm to a region close to the microtubule-organizing center in these cells. FAK and PYK-2 activation was blocked when lymphoblasts were pretreated with actin and tubulin cytoskeleton-interfering agents, indicating its cytoskeletal dependence. Our results demonstrate that interaction of the β2-integrin LFA-1 with its ligand intercellular adhesion molecule 1 induces remodeling of T lymphocyte morphology and activation and redistribution of the cytoplasmic tyrosine kinases FAK and PYK-2.

Tyrosine Phosphorylation of Selected Secretory Carrier Membrane Proteins, SCAMP1 and SCAMP3, and Association with the EGF Receptor

Secretory carrier membrane proteins (SCAMPs) are ubiquitously expressed proteins of post-Golgi vesicles. In the presence of the tyrosine phosphatase inhibitor vanadate, or after overexpression in Chinese hamster ovary (CHO) cells, SCAMP1 and SCAMP3 are phosphorylated selectively on tyrosine residue(s). Phosphorylation is reversible after vanadate washout in situ or when isolated SCAMP3 is incubated with the recombinant tyrosine phosphatase PTP1B. Vanadate also causes the partial accumulation of SCAMP3, but not SCAMP1, in “patches” at or near the cell surface. A search for SCAMP kinase activities has shown that SCAMPs 1 and 3, but not SCAMP2, are tyrosine phosphorylated in EGF-stimulated murine fibroblasts overexpressing the EGF receptor (EGFR). EGF catalyzes the progressive phosphorylation of the SCAMPs up to 1 h poststimulation and may enhance colocalization of the EGFR and SCAMP3 within the cell interior. EGF also induces SCAMP–EGFR association, as detected by coimmunoprecipitation, and phosphorylation of SCAMP3 is stimulated by the EGFR in vitro. These results suggest that phosphorylation of SCAMPs, either directly or indirectly, may be functionally linked to the internalization/down-regulation of the EGFR.

Serine and Threonine Phosphorylation of the Paxillin LIM Domains Regulates Paxillin Focal Adhesion Localization and Cell Adhesion to Fibronectin

We have previously shown that the LIM domains of paxillin operate as the focal adhesion (FA)-targeting motif of this protein. In the current study, we have identified the capacity of paxillin LIM2 and LIM3 to serve as binding sites for, and substrates of serine/threonine kinases. The activities of the LIM2- and LIM3-associated kinases were stimulated after adhesion of CHO.K1 cells to fibronectin; consequently, a role for LIM domain phosphorylation in regulating the subcellular localization of paxillin after adhesion to fibronectin was investigated. An avian paxillin-CHO.K1 model system was used to explore the role of paxillin phosphorylation in paxillin localization to FAs. We found that mutations of paxillin that mimicked LIM domain phosphorylation accelerated fibronectin-induced localization of paxillin to focal contacts. Further, blocking phosphorylation of the LIM domains reduced cell adhesion to fibronectin, whereas constitutive LIM domain phosphorylation significantly increased the capacity of cells to adhere to fibronectin. The potentiation of FA targeting and cell adhesion to fibronectin was specific to LIM domain phosphorylation as mutation of the amino-terminal tyrosine and serine residues of paxillin that are phosphorylated in response to fibronectin adhesion had no effect on the rate of FA localization or cell adhesion. This represents the first demonstration of the regulation of protein localization through LIM domain phosphorylation and suggests a novel mechanism of regulating LIM domain function. Additionally, these results provide the first evidence that paxillin contributes to “inside-out” integrin-mediated signal transduction.

Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo in Saccharomyces cerevisiae

Cyclophilin and FK506 binding protein (FKBP) accelerate cis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

Derepressed Hyphal Growth and Reduced Virulence in a VH1 Family-related Protein Phosphatase Mutant of the Human Pathogen Candida albicans

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.

Aberrant B cell receptor signaling from B29 (Igβ, CD79b) gene mutations of chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igβ, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing μ, κ, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with μ or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa

Factor VIIa (VIIa), the serine protease that initiates the coagulation pathways, is catalytically activated upon binding to its cell surface receptor and cofactor tissue factor (TF). This study provides a comprehensive analysis of the functional surface of VIIa by alanine scanning mutagenesis of 112 residues. Residue side chains were defined which contribute to TF binding and factor X hydrolysis. Energetically important binding contacts at the interface with TF were identified in the first epidermal growth factor domain of VIIa (Gln-64, Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306, Asp-309). The observed energetic defects are in good agreement with the corresponding residues in TF, suggesting that the VIIa light chain plays a prominent role in high affinity binding of cofactor. Mutation of protease domain interface residues indicated that TF allosterically influences the active site of VIIa. Stabilization of a labile zymogen to enzyme transition could explain the activating effect of TF on VIIa catalytic function. Residues important for factor X hydrolysis were found in three regions of the protease domain: (i) specificity determinants in the catalytic cleft and adjacent loops, (ii) an exosite near the TF binding site, and (iii) a large electronegative exosite which is in a position analogous to the basic exosite I of thrombin. TF regions involved in factor X activation are positioned on the same face of the TF·VIIa complex as the two exosites identified on the protease domain surface, providing evidence for an extended interaction of TF·VIIa with macromolecular substrate.