Nitration of profilin effects its interaction with poly (L-proline) and actin

Profilin from bovine spleen was nitrated with peroxynitrite; immunoblotting and spectrophotometric quantitation of nitrotyrosine residues suggested nitration of a single tyrosine residue in profilin with a stoichiometry of 0.6 mol of nitrotyrosine/mole of profilin. A decrease in the nitrotyrosine immunoreactivity of nitroprofilin during digestion with carboxypeptidase Y indicated that nitrotyrosine is located at the C-terminus of profilin. Nitroprofilin interaction with ligands such as phosphatidylinositol 4,5-bisphosphate, actin and poly (l-proline) was analyzed by monitoring the tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no significant difference in affinity of nitroprofilin to phosphatidylinositol 4,5-bisphosphate (K(d) of 4.8 +/- 0.5 muM for profilin, and K(d) of 5.7 +/- 0.6 muM for nitroprofilin), while poly (l-proline) binding studies revealed a twenty-fold increase in the affinity of profilin to poly (l-proline) upon nitration (K(d) of 21.8 +/- 1.7 muM for profilin, and K(d) of 1.1 +/- 0.1 muM for nitroprofilin). Actin polymerization studies involving pyrene-labeled actin indicated that profilin nitration inhibits the actin sequestering property of profilin. The critical actin monomer concentration (C(c)) was 150 and 250 nM in the presence of nitroprofilin and profilin, respectively. Thus, nitric oxide and free radicals produced under different conditions could alter the functions of profilin through nitration, such as its interaction with actin and poly (l-proline).     

Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.     

Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments

We have determined the absolute phosphate content of microtubule-associated proteins (MAPs) and established that phosphorylation inhibits the actin filament cross-linking activity of MAPs and both of the major MAP components, MAP-2 and tau. Similar results were obtained with actin from rabbit muscle, hog brain, and Acanthamoeba castellanii. We used the endogenous phosphatases and kinases in hog brain microtubule protein to modulate MAP phosphate level before isolating heat-stable MAPs. MAPs isolated directly from twice-cycled microtubule protein contain 7.1 +/- 0.1 (S.E.) mol of phosphate/300,000 g protein. After incubating microtubule protein without ATP, MAPs, had 4.9 +/- 0.6 phosphates. After incubating microtubule protein with 1 mM ATP and 5 microM cAMP in 2 mM EGTA, MAPs had 8.6 +/- 0.5 phosphates but there was also exchange of three more [32P]phosphates from gamma-labeled ATP for preexisting MAP phosphate. Incubation of microtubule protein with ATP and cAMP in 5 mM CaCl2 resulted in exchange but no net addition of phosphate to MAPs. We fractionated the MAP preparations by gel filtration and obtained MAP-2 with 4.3 to 7.5 and tau with 1.5 to 2.2 mol of phosphate/mol of protein depending on how we treated the microtubule protein prior to MAP isolation. The actin filament cross-linking activity of whole MAPs, MAP-2, and tau depended on the MAP-phosphate content. In all cases, phosphorylation of MAPs inhibited actin filament cross-linking activity. The concentration of high phosphate MAPs required to form a high viscosity solution with actin filaments was 2 to 4 times more than that of low phosphate. MAPs. During incubation of microtubule protein with [gamma-32P]ATP, only MAP peptides are labeled. Treatment of these MAPs with either acid or alkaline phosphatase removes phosphate mainly from MAP-2, with an increase in actin filament cross-linking activity. Thus, both MAP phosphorylation and the effect of phosphorylation on actin cross-linking activity of MAPs are reversible.     

Phosphorylation of spinophilin modulates its interaction with actin filaments

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission.     

The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin beta and profilin-actin gamma. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg2+, and can be crystallized. From the isolated profilin-actin complexes the beta- and gamma-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.     

Mechanism of the interaction of human platelet profilin with actin

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.     

Phosphorylation of profilin by ROCK1 regulates polyglutamine aggregation

Y-27632, an inhibitor of the Rho-associated kinase ROCK, is a therapeutic lead for Huntington disease (HD). The downstream targets that mediate its inhibitory effects on huntingtin (Htt) aggregation and toxicity are unknown. We have identified profilin, a small actin-binding factor that also interacts with Htt, as being a direct target of the ROCK1 isoform. The overexpression of profilin reduces the aggregation of polyglutamine-expanded Htt and androgen receptor (AR) peptides. This requires profilin's G-actin binding activity and its direct interaction with Htt, which are both inhibited by the ROCK1-mediated phosphorylation of profilin at Ser-137. Y-27632 blocks the phosphorylation of profilin in HEK293 cells and primary neurons, which maintains profilin in an active state. The knockdown of profilin blocks the inhibitory effect of Y-27632 on both AR and Htt aggregation. A signaling pathway from ROCK1 to profilin thus controls polyglutamine protein aggregation and is targeted by a promising therapeutic lead for HD.     

Isolation of profilin from embryonic chicken skeletal muscle and evaluation of its interaction with different actin isoforms

An actin-binding protein of 16 kDa was isolated from embryonic chicken skeletal muscle. The protein had the same properties as profilin, exhibited a much higher affinity for cytoskeletal (beta- and gamma-) actins than for sarcomeric (alpha-) actin in the embryonic muscle, and inhibited the polymerization of beta- and gamma-actins more efficiently in a physiological salt solution. These results indicate that the assembly of cytoskeletal and sarcomeric actins is regulated differently by profilin in the developing skeletal muscle, and that the former may not be involved in myofibril assembly.     

Phosphorylation of Numb regulates its interaction with the clathrin-associated adaptor AP-2

Numb is thought to participate in clathrin-dependent endocytosis by directly interacting with the clathrin-associated adaptor complex AP-2, although the underlying mechanisms are unknown. Numb is also known to be phosphorylated at Ser(264)in vitro and in vivo. Here, we found that Numb is phosphorylated in vitro by Ca(2+)/calmodulin-dependent protein kinase I on Ser(283). This phosphorylation was also observed in transfected COS-7 cells, indicating its physiological relevance. Pull-down experiments showed that the phosphorylation of Numb impaired its binding to the AP-2 complex and simultaneously recruited 14-3-3 proteins in vitro. Based on experiments using Numb mutants, both the initial phosphorylation of Ser(264) and the subsequent phosphorylation of Ser(283) are sufficient to abolish the binding of Numb to AP-2 and to promote the interaction with 14-3-3 protein. These findings suggest a novel mechanism for the regulation of Numb-mediated endocytosis, namely through direct phosphorylation.     

Phosphorylation of NuSAP by Cdk1 regulates its interaction with microtubules in mitosis

Cell division in eukaryotes depends on a fine control of the dynamic changes of microtubules. Nucleolar and spindle-associated protein (NuSAP) is a microtubule-binding and -bundling protein essential for the integrity of the anaphase spindle and cell division. NuSAP contains two consensus cdk phosphorylation sites in its microtubule-binding domain. Here we show NuSAP is phosphorylated by cdk1 in early mitosis. This phosphorylation inhibits the binding of NuSAP to microtubules. During metaphase-to anaphase transition, NuSAP is dephosphorylated to promote spindle midzone formation and cell cycle progression. Expression of cdk1 phosphorylation-null mutant causes extensive bundling of microtubules in the prometaphase spindle. Our results suggest that phosphorylation and dephosphorylation of NuSAP during progression of mitosis regulate spindle organization through modulation of the dynamics of microtubules.     

Phosphorylation of grb10 regulates its interaction with 14-3-3

Grb10 is a member of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. Grb10 expression levels can influence Akt activity, and Grb10 may act as an adapter involved in the relocalization of Akt to the cell membrane. Here we identified 14-3-3 as a binding partner of Grb10 by employing a yeast two-hybrid screen. The 14-3-3.Grb10 interaction requires phosphorylation of Grb10, and only the phosphorylated form of Grb10 co-immunoprecipitates with endogenous 14-3-3. We could identify a putative phosphorylation site in Grb10, which is located in a classical 14-3-3 binding motif, RSVSEN. Mutation of this site in Grb10 diminished binding to 14-3-3. Thus, Grb10 exists in two different states of phosphorylation and complexes with 14-3-3 when phosphorylated on serine 428. We provide evidence that Akt directly binds Grb10 and is able to phosphorylate Grb10 in an in vitro kinase assay. Based on these findings, we propose a regulatory circuitry involving a phosphorylation-regulated complex formation of Grb10 with 14-3-3 and Akt.     

Phosphorylation of the amino-terminal region of X11L regulates its interaction with APP

X11-like (X11L) is neuronal adaptor protein that interacts with the amyloid β-protein precursor (APP) and regulates its metabolism. The phosphotyrosine interaction/binding (PI/PTB) domain of X11L interacts with the cytoplasmic region of APP695. We found that X11L–APP interaction is enhanced in osmotically stressed cells and X11L modification is required for the enhancement. Amino acids 221–250 (X11L^221–250) are required for the enhanced association with APP in osmotically stressed cells; this motif is 118 amino acids closer to the amino-terminal end of the protein than the PI/PTB domain (amino acids 368–555). We identified two phosphorylatable seryl residues, Ser236 and Ser238, in X11L^221–250 and alanyl substitution of either seryl residue diminished the enhanced association with APP. In brain Ser238 was found to be phosphorylated and phosphorylation of X11L was required for the interaction of X11L and APP. Both seryl residues in X11L^221–250 are conserved in neuronal X11, but not in X11L2, a non-neuronal X11 family member that did not exhibit enhanced APP association in osmotically stressed cells. These findings indicate that the region of X11L that regulates association with APP is located outside of, and amino-terminal to, the PI/PTB domain. Modification of this regulatory region may alter the conformation of the PI/PTB domain to modulate APP binding.     

Class-specific interaction of profilin and ADF isovariants with actin in the regulation of plant development

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein-protein interactions that are essential to the normal regulation of plant growth and development.     

Phosphorylation of stargazin by protein kinase A regulates its interaction with PSD-95.

Stargazin is the first transmembrane protein known to associate with AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) glutamate receptors (AMPARs) and regulate their synaptic targeting by two distinct mechanisms, specifically via delivery of AMPARs to the surface membrane and synaptic targeting of these receptors by binding to PSD-95/SAP-90 and related PDZ proteins. However, it is not known whether and how this stargazin-mediated synaptic targeting of AMPARs is regulated. Stargazin interacts with the PDZ domains of PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus contains a consensus sequence for phosphorylation by cAMP-dependent protein kinase A (PKA). Phosphorylation site-specific stargazin antibodies reveal that the stargazin C terminus is phosphorylated at the Thr-321 residue in heterologous cells and in vivo. Stargazin phosphorylation is enhanced by the catalytic subunit of PKA. Mutations mimicking stargazin phosphorylation (T321E and T321D) lead to elimination of yeast two-hybrid interactions, in vitro coimmunoprecipitation, and coclustering between stargazin and PSD-95. Phosphorylated stargazin shows a selective loss of coimmunoprecipitation with PSD-95 in heterologous cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phosphorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs.     

Properties of actin from the fission yeast Schizosaccharomyces pombe and interaction with fission yeast profilin

The fission yeast Schizosaccharomyces pombe serves as a model system for studying role of actin cytoskeleton, since it has simple actin cytoskeletons and is genetically tractable. In contrast, biochemical approaches using this organism are still developing; fission yeast actin has so far not been isolated in its native form and characterized, and therefore, biochemical assays of fission yeast actin-binding proteins (ABPs) or myosin have been performed using rabbit skeletal muscle actin that may interact with the fission yeast ABPs in a manner different from fission yeast actin. Here, we report a novel method for isolating functionally active actin from fission yeast cells. The highly purified fission yeast actin polymerized with kinetics somewhat different from those of muscle actin and forms filaments that are structurally indistinguishable from skeletal muscle actin filaments. The fission yeast actin was a significantly weaker activator of Mg(2+)-ATPase of HMM of skeletal muscle myosin than muscle actin. The fission yeast profilin Cdc3 suppressed polymerization of fission yeast actin more effectively than that of muscle actin and showed an affinity for fission yeast actin higher than for muscle actin. The establishment of purification of fission yeast actin will enable reconstruction of physiologically relevant interactions between the actin and fission yeast ABPs or myosins and contribute to clarification of function of actin cytoskeleton in various cellular activities.     

Poly(L-proline)-binding proteins from chick embryos are a profilin and a profilactin

Two poly(L-proline)-binding proteins (PBP-1 and PBP-2) were purified from chick embryos by using a poly(L-proline)-agarose column. PBP-1 was composed of two different polypeptides (molecular masses of 42 kDa and 15 kDa). The molar ratio of the two proteins in the complex was 1:1. The other poly(L-proline)-binding protein, PBP-2, was the 15-kDa protein itself. The 42-kDa protein was confirmed to be an actin from the amino acid composition, by immunochemical evidence and by its ability to self-polymerize. In addition, the 42 + 15-kDa protein complex (PBP-1) inhibited DNase I, just as a monomeric actin did. The amino acid composition of the 15-kDa protein was similar to that of mammalian profilin and it inhibited the salt-induced polymerization of rabbit skeletal muscle actin. Therefore, we conclude that the two poly(L-proline)-binding proteins from the chick embryo are a profilactin and a profilin in chick embryo. The ability of profilactin to bind poly(L-proline) must be due to profilin itself, because the profilin has a greater affinity for poly(L-proline) than does profilactin. Additionally, both the monomeric and filamentous actin from rabbit skeletal muscle have no affinity for poly(L-proline).     

Molecular packing in profilin: actin crystals and its implications

Analysis of profilin: actin crystals reveals an extensive intermolecular network, rather than a discrete "monomeric complex", comprising stacked actin ribbons held in place by columns of profilin molecules, wedged in between neighboring actin subunits and running perpendicular to the ribbons. Comparison with data from electron microscopy, X-ray diffraction, spectroscopy, and biochemistry of actin suggests that a simple transformation relates the ribbon to f-actin. The crystals exhibit unusual polymorphic properties, which strengthens the view that movements within the actin monomer are important for force generation.     

Phosphorylation of LRP1 regulates the interaction with Fe65

Neuronal Fe65 is a central adapter for the intracellular protein network of Alzheimer's disease related amyloid precursor protein (APP). It contains a unique tandem array of phosphotyrosine-binding (PTB) domains that recognize NPXY internalization motifs present in the intracellular domains of APP (AICD) and the low-density lipoprotein receptor-related protein LRP1 (LICD). The ternary APP/Fe65/LRP1 complex is an important mediator of APP processing and affects β-amyloid peptide production. Here we dissect by biochemical and biophysical methods the direct interactions within the ternary complex and reveal a phosphorylation-dependent insulin receptor substrate (IRS-) like interaction of the distal NPVY(4507) motif of LICD with Fe65-PTB1.     

Plant prolyl hydroxylase recognizes poly(L-proline) II helix

Substrate specificity of a prolyl hydroxylase from Vinca rosea suspension-cultured cells was studied using synthetic oligo(L-proline)s and their t-butyloxycarbonyl derivatives (Pron and Boc-Pron; n = 2-8) as peptidyl substrates. All peptides with a residual number of 5 or greater served as substrates in the enzyme reaction at 30 degrees C, after the preincubation of the enzyme and peptides at 0 degrees C prior to addition of cofactors and cosubstrate. Under the same conditions, the hydroxylation of Pro5 reached a plateau within 10 min, but that of Boc-Pro8 and poly(L-proline) increased linearly up to 40 min. If the preincubation temperature was raised to 30 degrees C, only Pro5 among the peptides was unable to serve as a substrate. The optimum temperature of the enzyme was 30 degrees C toward Boc-Pro8 and poly(L-proline) but it decreased to 15 degrees C using Pro5. These data suggest that the enzyme can bind Pro5 only at low temperature. Poly(L-proline) and Boc-Pron (n greater than or equal to 5) in aqueous solution are known to have a left-handed helical structure (poly(L-proline) II helix). Moreover, Pro5 was indicated as forming this helix below 10 degrees C. Accordingly, the enzyme recognizes the poly(L-proline) II helix, that is, the secondary structure of a substrate rather than the primary structure.     

Chirality responsive helical poly(phenylacetylene) bearing L-proline pendants

A novel, optically active, cis-transoidal poly(phenylacetylene) bearing an L-proline residue as the pendant group (poly-1) was prepared by the polymerization of the corresponding monomer using a rhodium catalyst in water, and its chiroptical property was investigated using circular dichroism spectroscopy. Poly-1 showed intense Cotton effects in the UV-visible region of the polymer backbone in water, resulting from the prevailing one-handed helical conformation induced by the covalent-bonded chiral L-proline pendants and exhibited a unique helix-sense inversion in response to external, achiral, and chiral stimuli, such as the solvent and interactions with chiral small molecules. We found that poly-1 could enantioselectively trap 1,1'-2-binaphthol within its hydrophobic helical cavity inside the polymer in aqueous media and underwent an inversion of its helical sense in the presence of one of the enantiomers. The effect of the optical purity of 1,1'-2-binaphthol on the chiroptical properties of poly-1 was also investigated.