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• W2593236198 abstract "Pathogenic Yersinia bacteria cause a range of human diseases. To modulate and evade host immune systems, these yersiniae inject effector proteins into host macrophages. One such protein, the serine/threonine kinase YopO (YpkA in Yersinia pestis), uses monomeric actin as bait to recruit and phosphorylate host actin polymerization-regulating proteins, including the actin-severing protein gelsolin, to disrupt actin filaments and thus impair phagocytosis. However, the YopO phosphorylation sites on gelsolin and the consequences of YopO-mediated phosphorylation on actin remodeling have yet to be established. Here we determined the effects of YopO-mediated phosphorylation on gelsolin and identified its phosphorylation sites by mass spectrometry. YopO phosphorylated gelsolin in the linker region between gelsolin homology domains G3 and G4, which, in the absence of calcium, are compacted but adopt an open conformation in the presence of calcium, enabling actin binding and severing. Using phosphomimetic and phosphodeletion gelsolin mutants, we found that YopO-mediated phosphorylation partially mimics calcium-dependent activation of gelsolin, potentially contributing to a reduction in filamentous actin and altered actin dynamics in phagocytic cells. In summary, this work represents the first report of the functional outcome of serine/threonine phosphorylation in gelsolin regulation and provides critical insight into how YopO disrupts normal gelsolin function to alter host actin dynamics and thus cripple phagocytosis. Pathogenic Yersinia bacteria cause a range of human diseases. To modulate and evade host immune systems, these yersiniae inject effector proteins into host macrophages. One such protein, the serine/threonine kinase YopO (YpkA in Yersinia pestis), uses monomeric actin as bait to recruit and phosphorylate host actin polymerization-regulating proteins, including the actin-severing protein gelsolin, to disrupt actin filaments and thus impair phagocytosis. However, the YopO phosphorylation sites on gelsolin and the consequences of YopO-mediated phosphorylation on actin remodeling have yet to be established. Here we determined the effects of YopO-mediated phosphorylation on gelsolin and identified its phosphorylation sites by mass spectrometry. YopO phosphorylated gelsolin in the linker region between gelsolin homology domains G3 and G4, which, in the absence of calcium, are compacted but adopt an open conformation in the presence of calcium, enabling actin binding and severing. Using phosphomimetic and phosphodeletion gelsolin mutants, we found that YopO-mediated phosphorylation partially mimics calcium-dependent activation of gelsolin, potentially contributing to a reduction in filamentous actin and altered actin dynamics in phagocytic cells. In summary, this work represents the first report of the functional outcome of serine/threonine phosphorylation in gelsolin regulation and provides critical insight into how YopO disrupts normal gelsolin function to alter host actin dynamics and thus cripple phagocytosis. Gelsolin remodels actin organization through Ca2+-dependent severing and capping of actin filaments (1Nag S. Larsson M. Robinson R.C. Burtnick L.D. Gelsolin: the tail of a molecular gymnast.Cytoskeleton. 2013; 70: 360-384Crossref Scopus (149) Google Scholar). Gelsolin also exists in the blood plasma as an 83-kDa protein, forming part of the extracellular actin-scavenging system. Intracellularly, it is present as an 81-kDa protein in a wide range of cell types (1Nag S. Larsson M. Robinson R.C. Burtnick L.D. Gelsolin: the tail of a molecular gymnast.Cytoskeleton. 2013; 70: 360-384Crossref Scopus (149) Google Scholar). It is implicated in various cellular processes, including cell motility, phagocytosis, signaling, apoptosis, cancer, and platelet activation, and it has roles in a number of diseases (2Li G.H. Arora P.D. Chen Y. McCulloch C.A. Liu P. Multifunctional roles of gelsolin in health and diseases.Med. Res. Rev. 2012; 32: 999-1025Crossref PubMed Scopus (171) Google Scholar). In familial amyloidosis of the Finnish type, point mutations in gelsolin increase its susceptibility to proteolysis, resulting in amyloid deposits in the eyes, skin, and nerves (3Maury C.P. Gelsolin-related amyloidosis. Identification of the amyloid protein in Finnish hereditary amyloidosis as a fragment of variant gelsolin.J. Clin. Invest. 1991; 87: 1195-1199Crossref PubMed Scopus (75) Google Scholar). High expression levels of gelsolin correlate with tumor size, poor patient prognosis, and increased invasive capacity in a range of cancers (4Huang B. Deng S. Loo S.Y. Datta A. Yap Y.L. Yan B. Ooi C.H. Dinh T.D. Zhuo J. Tochhawng L. Gopinadhan S. Jegadeesan T. Tan P. Salto-Tellez M. Yong W.P. et al.Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma.Oncotarget. 2016; 7: 25391-25407Crossref PubMed Scopus (10) Google Scholar). Gelsolin has well characterized roles in cell motility and the immune system. Gelsolin-null fibroblasts have pronounced actin stress fibers and migrate slower than the wild-type cells due to aberrant severing and remodeling of actin filaments (5Witke W. Sharpe A.H. Hartwig J.H. Azuma T. Stossel T.P. Kwiatkowski D.J. Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin.Cell. 1995; 81: 41-51Abstract Full Text PDF PubMed Scopus (379) Google Scholar), whereas gelsolin-null mice have multiple defects in morphology and motility in various cell types, including neutrophils, platelets, osteoclasts, and dermal fibroblasts (5Witke W. Sharpe A.H. Hartwig J.H. Azuma T. Stossel T.P. Kwiatkowski D.J. Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin.Cell. 1995; 81: 41-51Abstract Full Text PDF PubMed Scopus (379) Google Scholar). Gelsolin is involved in Fc-γ receptor (FcγR) 6The abbreviations used are: FcγRFc-γ receptorPIP2phosphatidylinositol 4,5-bisphosphateGDIguanidine nucleotide dissociation inhibitorPDBProtein Data BankBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolTMtriple mutationDMdouble mutationMDmolecular dynamics. and integrin-mediated phagocytosis and is enriched around the phagosome, particularly in IgG-mediated phagocytosis (6Serrander L. Skarman P. Rasmussen B. Witke W. Lew D.P. Krause K.H. Stendahl O. Nüsse O. Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils.J. Immunol. 2000; 165: 2451-2457Crossref PubMed Scopus (70) Google Scholar), and as such gelsolin-null neutrophils are impaired in FcγR-mediated phagocytosis, whereas gelsolin-null fibroblasts exhibit defective binding and phagocytosis (6Serrander L. Skarman P. Rasmussen B. Witke W. Lew D.P. Krause K.H. Stendahl O. Nüsse O. Selective inhibition of IgG-mediated phagocytosis in gelsolin-deficient murine neutrophils.J. Immunol. 2000; 165: 2451-2457Crossref PubMed Scopus (70) Google Scholar). Microbicidal activities of phagocytic cells require chemotaxis and phagocytosis, which involve gelsolin-dependent actin remodeling (7Groves E. Dart A.E. Covarelli V. Caron E. Molecular mechanisms of phagocytic uptake in mammalian cells.Cell. Mol. Life Sci. 2008; 65: 1957-1976Crossref PubMed Scopus (170) Google Scholar). Fc-γ receptor phosphatidylinositol 4,5-bisphosphate guanidine nucleotide dissociation inhibitor Protein Data Bank 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol triple mutation double mutation molecular dynamics. Gelsolin comprises six gelsolin homology domains (G1–G6), which, in the absence of calcium, exist in a compact globular arrangement (8Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In the presence of micromolar concentrations of calcium, gelsolin undergoes large conformational changes to adopt an open conformation in which the 47-residue-long linker between G3 and G4 extends, allowing the two halves (G1-G3 and G4-G6) to bind the barbed ends of actin filaments, exposing the major actin-binding surfaces on G1, G2-G3, and G4 (9Robinson R.C. Mejillano M. Le V.P. Burtnick L.D. Yin H.L. Choe S. Domain movement in gelsolin: a calcium-activated switch.Science. 1999; 286: 1939-1942Crossref PubMed Scopus (134) Google Scholar, 10Choe H. Burtnick L.D. Mejillano M. Yin H.L. Robinson R.C. Choe S. The calcium activation of gelsolin: insights from the 3Å structure of the G4–G6/Actin complex.J. Mol. Biol. 2002; 324: 691-702Crossref PubMed Scopus (114) Google Scholar). Activated gelsolin binds to the side of an actin filament and severs that filament into two smaller polymers while capping the barbed end of one-half of the severed filament. The binding of polyphosphoinositides (PPIs) to gelsolin (particularly PIP2) uncaps gelsolin from the barbed end to re-enable growth of the actin filament (11Janmey P.A. Stossel T.P. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate.Nature. 1987; 325: 362-364Crossref PubMed Scopus (494) Google Scholar). Besides regulation by calcium and PIP2, gelsolin can also be regulated by pH, proteolysis, and phosphorylation. Gelsolin undergoes calcium-independent activation below pH 6 (12Lamb J.A. Allen P.G. Tuan B.Y. Janmey P.A. Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement.J. Biol. Chem. 1993; 268: 8999-9004Abstract Full Text PDF PubMed Google Scholar). Caspase-3, a critical mediator of apoptosis, cleaves gelsolin in the linker region between domains 3 and 4, giving rise to an N-terminal gelsolin fragment (residues 1–352) that is pro-apoptotic (13Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis.Science. 1997; 278: 294-298Crossref PubMed Scopus (1039) Google Scholar). Gelsolin is phosphorylated by the tyrosine kinase c-Src primarily at Tyr-438 in the domain G4. Artificial phosphorylation of plasma gelsolin at its N terminus removes its dependence on calcium activation (14Takiguchi K. Yamashiro-Matsumura S. Matsumura F. Artificial phosphorylation removes gelsolin's dependence on calcium.Cell Struct. Funct. 2000; 25: 57-65Crossref PubMed Scopus (4) Google Scholar). In osteoclasts, proline-rich tyrosine kinase 2 (PYK2) phosphorylates gelsolin on tyrosine residues, reducing gelsolin-actin interactions while increasing the association of gelsolin with PIP2 (15Wang Q. Xie Y. Du Q.S. Wu X.J. Feng X. Mei L. McDonald J.M. Xiong W.C. Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin.J. Cell Biol. 2003; 160: 565-575Crossref PubMed Scopus (99) Google Scholar). Our recent work has shown that YopO kinase from Yersinia phosphorylates human gelsolin through a mechanism that involves the formation of a ternary complex with actin (16Lee W.L. Grimes J.M. Robinson R.C. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.Nat. Struct. Mol. Biol. 2015; 22: 248-255Crossref PubMed Scopus (38) Google Scholar). Yersinia bacteria cause a range of human diseases, including gastrointestinal syndromes (Yersinia enterocolitica), Far East scarlet-like fever (Yersinia pseudotuberculosis), and the plague (Yersinia pestis). Pathogenic Yersinia species use a type III secretion system to inject effector proteins (Yops) into host macrophages. These comprise six proteins, YopO/YpkA, YopH, YopM, YopE, YopT, and YopJ, that collectively inhibit phagocytosis and reorient the host immune responses (17Cornelis G.R. Boland A. Boyd A.P. Geuijen C. Iriarte M. Neyt C. Sory M.P. Stainier I. The virulence plasmid of Yersinia, an antihost genome.Microbiol. Mol. Biol. Rev. 1998; 62: 1315-1352Crossref PubMed Google Scholar). YopO, also known as YpkA in Y. enterocolitica, harbors a serine/threonine kinase domain (18Galyov E.E. Håkansson S. Forsberg A. Wolf-Watz H. A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant.Nature. 1993; 361: 730-732Crossref PubMed Scopus (257) Google Scholar) and a guanidine nucleotide dissociation inhibitor (GDI)-like domain (19Prehna G. Ivanov M.I. Bliska J.B. Stebbins C.E. Yersinia virulence depends on mimicry of host Rho-family nucleotide dissociation inhibitors.Cell. 2006; 126: 869-880Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The kinase activity of YopO is activated in the host cytosol upon binding to actin (20Trasak C. Zenner G. Vogel A. Yüksekdag G. Rost R. Haase I. Fischer M. Israel L. Imhof A. Linder S. Schleicher M. Aepfelbacher M. Yersinia protein kinase YopO is activated by a novel G-actin binding process.J. Biol. Chem. 2007; 282: 2268-2277Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Actin becomes sandwiched between the serine/threonine kinase domain and the GDI-like domain, which stabilizes the kinase active site in an active conformation (16Lee W.L. Grimes J.M. Robinson R.C. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.Nat. Struct. Mol. Biol. 2015; 22: 248-255Crossref PubMed Scopus (38) Google Scholar). Actin-binding proteins are recruited to YopO-actin through the bound actin to form ternary complexes, some of which are phosphorylated by YopO, including Ena/VASP family proteins, DIAPH1, WASP, and gelsolin (16Lee W.L. Grimes J.M. Robinson R.C. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.Nat. Struct. Mol. Biol. 2015; 22: 248-255Crossref PubMed Scopus (38) Google Scholar). However, the phosphorylation sites on the phosphorylated substrates and their consequences on actin remodeling have yet to be examined. In this study, we determined the functional implications of gelsolin phosphorylation by YopO. We identified the phosphorylation sites on gelsolin by mass spectrometry and validated these sites by in vitro phosphorylation assays. Molecular dynamics simulations on the modeled phosphorylated gelsolin indicate that phosphorylation by YopO contributes to the instability of inactive gelsolin. Furthermore, actin polymerization assays of phosphomimetic mutants of gelsolin confirm that phosphorylation by YopO confers on gelsolin Ca2+-independent activation. Thus, these findings suggest how YopO may circumvent the native calcium-signaling pathways for gelsolin activation to manipulate the host actin cytoskeleton and resist phagocytosis. To identify the YopO phosphorylation sites on gelsolin, we carried out an in vitro phosphorylation assay of gelsolin by YopO in the presence of actin and analyzed the modifications by LC-MS/MS. Phosphorylation sites were identified at Ser-381, Ser-384, and Ser-385 (numbering corresponds to plasma gelsolin). These residues lie in the linker region between the gelsolin domains G3 and G4 (Fig. 1A and supplemental Fig. S1). To validate these sites, phospho-deletion mutants of gelsolin, in which the respective serine residues were mutated to alanine, were generated (supplemental Table S1) and tested for radioactive γ-32P incorporation in the presence of YopO and Sf9 actin. In comparison with wild-type (WT) gelsolin, the triple mutant TM (381AYLAA385) displayed the least phosphorylation, and the double mutant DM3 (381AYLSA385) had a similar level of phosphorylation as TM (Fig. 1B and supplemental Fig. S2). Both TM and DM3 possess mutations at positions 381 and 385, indicating that YopO readily phosphorylates these residues in WT gelsolin. The double mutant DM2 (381SYLAA385) showed a significant reduction in phosphorylation, whereas DM1 (381AYLAS385) had only a slight attenuation. These data suggest that Ser-385 is the major phosphorylation site, based on the reduction in incorporation of radioactivity observed for the mutants TM and DM3, whereas Ser-381 and Ser-384 are minor phosphorylation sites. Sequence alignments of gelsolin across species revealed the conservation of Ser-385 across human, mouse, rat, pig, horse, and bovine but not in chicken (Fig. 1C). Chickens are rarely transmission hosts for Yersinia (21Heroven A.K. Dersch P. Coregulation of host-adapted metabolism and virulence by pathogenic yersiniae.Front. Cell. Infect. Microbiol. 2014; 4: 146Crossref PubMed Scopus (44) Google Scholar). To obtain insight into the structural implications that could arise from phosphorylation at Ser-381 and Ser-385, we modeled their positions in the crystal structure of full-length inactive gelsolin (Fig. 2A) (PDB code 3FFN (22Nag S. Ma Q. Wang H. Chumnarnsilpa S. Lee W.L. Larsson M. Kannan B. Hernandez-Valladares M. Burtnick L.D. Robinson R.C. Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13713-13718Crossref PubMed Scopus (90) Google Scholar)). The residues that lie in the vicinity of these serines, as derived from the inactive structure of full-length human cytoplasmic gelsolin (PDB code 3FFN), are tabulated in supplemental Table S2. Ser-381 packs against G3, lying in close proximity to the negatively charged Glu-358 and bulky Trp-369 on G3 at distances of 3.8 and 4.7 Å, respectively. Ser-381 also lies in close proximity to the negatively charged residues Asp-371 and Glu-374 on the G3-G4 linker, at distances of 2.9 and 4.2 Å, respectively. Likewise, Ser-385 packs against G1 and lies in close proximity to the charged residues Lys-72 (4.3 Å) and Asp-84 (6.3 Å) and hydrophobic residues Tyr-133 and Val-74 in G1. We hypothesized that the introduction of negative charges by phosphorylation, coupled with steric hindrance from the much bulkier phosphate groups, may induce repulsion of G1 and G3. To test this hypothesis, we carried out molecular dynamics simulations with calcium-free WT gelsolin and the phosphorylated model (based on PDB code 3FFN) to verify that phosphorylation of these sites results in instability to the compact form of calcium-free inactive gelsolin. Although WT gelsolin remained stable for 250 ns following a 50-ns equilibration, gelsolin that has been phosphorylated at Ser-381, Ser-384, and Ser-385 consistently increased its radius of gyration (Rg) (Fig. 2B), and the distance between the centers of the masses of G1–G3 and G4–G6 during this time period (Fig. 2C), indicating a destabilizing effect by phosphorylation on the inactive conformation. Size-exclusion chromatography coupled with multiangle light scattering experiments confirmed that the phosphomimetic triple mutant (PM5, S381D/S384D/S385D), where the serines were mutated to aspartic acids to mimic phosphorylation, had higher R(avg) (geometric and hydrodynamic radius) than WT gelsolin, supporting the MD simulation data that predicted that the phosphorylated form of gelsolin adopts a more extended conformation than the WT (supplemental Table S3). To test the effect of phosphorylation on the gelsolin-actin interaction, in vitro-phosphorylated gelsolin was tested for its ability to sever actin filaments via a sedimentation assay. Under calcium-free conditions, gelsolin, phosphorylated by YopO WT, was active in severing actin filaments, as judged by the appearance of actin in the soluble fraction, compared with the non-phosphorylated gelsolin, across a range of gelsolin concentrations (Fig. 3A). In the presence of calcium (1.0 mm), both phosphorylated and non-phosphorylated gelsolin was observed to sever actin to a similar extent as revealed by comparing the ratios of actin in the soluble versus pellet fractions (Ca WT YopO versus Ca KD YopO) (Fig. 3B). In an attempt to validate these results, a more extensive set of phosphomimetic mutants of gelsolin were created, where the serines were mutated to aspartic acid as follows: PM1 (S381D/S385D); PM2 (S381D); PM3 (S385D); PM4 (S384D); and PM5 (S381D/S384D/S385D). We measured the decrease in pyrenyl fluorescence of F-actin to compare the activities of WT gelsolin and phosphomimetic mutants on the depolymerization of actin. At saturating calcium concentrations (1.0 mm), both WT and the phosphomimetic mutants exhibit similar depolymerizing kinetics (Fig. 4). However, in calcium-free conditions (1.5 mm EGTA), WT gelsolin remained inactive, yet the triple phosphomimetic mutant (PM5, S381D/S384D/S385D) displayed pronounced actin depolymerization activity.Figure 4Pyrene actin depolymerization assay with gelsolin phosphomimetic mutants in EGTA conditions. Polymerized F-actin (12 μm) was incubated with different gelsolin mutants (6 μm) in 1.5 mm EGTA or 1.0 mm calcium. The loss of pyrenyl fluorescence was measured. In EGTA, the triple mutant PM5 is more active at severing actin filaments, compared with WT and the other mutants. In calcium, phosphomimetic mutants and WT depolymerize actin filaments to similar extents.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Following this, the severing activities of WT gelsolin and PM5 were compared in an actin sedimentation assay (Fig. 5A). At saturating calcium levels (1.0 mm), PM5 severed and distributed actin into a soluble fraction similar to WT gelsolin, across a range of protein concentrations. However, in the absence of calcium, although WT gelsolin did not sever actin significantly, a substantial decrease in the pelletable actin fraction and a corresponding increase of actin in the supernatant were observed for the mutant PM5 (S381D/S384D/S385D) across the tested concentrations of gelsolin. Furthermore, PM5 appears to partition into the pellet fraction and co-sediment with F-actin, which may suggest that PM5 (in EGTA) preferentially severs and caps F-actin over sequestering G-actin. A slight redistribution of actin into the soluble fraction was detected with WT gelsolin in calcium-free conditions, possibly due to the residual activity of gelsolin. The proportion of actin in the pellet or soluble fraction in the presence of different concentrations of gelsolin is plotted in Fig. 5B. These data demonstrate that the incorporation of acidic residues into the linker region of gelsolin either reduces or removes its calcium requirement for activity. These data prompted us to further investigate the activities of the phosphomimetic mutants at different calcium concentrations (Fig. 6). A pyrene actin depolymerization assay was carried out against different calcium concentrations, in which the pyrene fluorescence was measured after 3 h upon addition of the various gelsolin constructs to F-actin. The value along the y axis is the measure of the fraction of F-actin remaining in the sample upon depolymerization by gelsolin. The y axis represents the percentage of actin that remains as F-actin following depolymerization by gelsolin. A fit to the data with the Hill equation (Fig. 6) yields Kd values for the calcium requirement for gelsolin activity of 0.37 μm for WT, 0.23 μm for PM1, 0.33 μm for PM2, 0.27 μm for PM3, 0.36 μm for PM4, and 0.11 μm for PM5, suggesting that PM5 (S381D/S384D/S385D) and PM1 (S381D/S385D) were substantially more active at depolymerizing actin than WT or other mutants at lower calcium concentrations. At intermediate concentrations of calcium, there is a small but significant increase in activity for PM3. At calcium concentrations above 0.7 μm, the activities of WT and phosphomimetic mutants were indistinguishable, agreeing with the depolymerization kinetics (Fig. 4). These biochemical results, together with structural modeling, suggest that the introduction of negative charges and the steric hindrance destabilizes the inactive closed conformation of gelsolin, paralleling calcium activation, where the binding of calcium destabilizes the inactive conformation. Because gelsolin is phosphorylated by YopO in its active actin-bound form, we hypothesize that the phosphorylated gelsolin will not be able to revert to the inactive conformation, thus remaining constitutively active. Next, we compared the actin nucleation activity of the phosphomimetic mutants in comparison with WT gelsolin (Fig. 7). In the absence of profilin, gelsolin nucleates actin filament formation by binding to two actin monomers to form an actin filament nucleus (23Janmey P.A. Chaponnier C. Lind S.E. Zaner K.S. Stossel T.P. Yin H.L. Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking.Biochemistry. 1985; 24: 3714-3723Crossref PubMed Scopus (154) Google Scholar). The actin filament nucleation properties of the WT and mutants were examined via a pyrene-actin polymerization assay at a gelsolin to actin ratio (1:40) in the presence of Ca2+ (0.1 mm). Ca2+ concentrations above 1 μm are required for gelsolin to bind to two actin monomers via its two halves G1–G3 and G4–G6 to form a stable actin nucleus (8Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Although WT gelsolin eliminated the lag phase and increased the initial rate of polymerization, four of the five phosphomimetic mutants exhibited reductions in nucleation activity. Nucleation was most strongly attenuated in PM5 (S381D/S384D/S385D) followed by the PM1 (S381D/S385D), PM3 (S385D), and PM2 (S381D). PM4 (S384D) was indistinguishable from WT. These data suggest that the substitution of the phosphorylation sites by aspartic acid, which partially mimics phosphorylation, lowers the in vitro nucleation activity, possibly by inducing a change in the G3–G4 linker to a conformation that is suboptimal for forming a stable actin nucleus with G1–G3 and G4–G6. Previously, we demonstrated that YopO uses actin as bait to recruit and phosphorylate actin-filament-regulating proteins (16Lee W.L. Grimes J.M. Robinson R.C. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.Nat. Struct. Mol. Biol. 2015; 22: 248-255Crossref PubMed Scopus (38) Google Scholar). YopO binds to subdomain 4 of actin, leaving the common binding pocket between subdomains 1 and 3 of actin available, a region of interaction for the majority of actin-binding proteins. A subset of actin filament-regulating proteins, those that are sufficiently elongated to span from the actin-binding site to the kinase catalytic cleft and can present suitable residues, are phosphorylated by YopO, including gelsolin, mDia1, VASP, EVL, and WASP (16Lee W.L. Grimes J.M. Robinson R.C. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.Nat. Struct. Mol. Biol. 2015; 22: 248-255Crossref PubMed Scopus (38) Google Scholar, 25Lee W.L. Singaravelu P. Wee S. Xue B. Ang K.C. Gunaratne J. Grimes J.M. Swaminathan K. Robinson R.C. Mechanisms of Yersinia YopO kinase substrate specificity. Mechanisms of Yersinia YopO kinase substrate specificity.Sci. Rep. 2017; 739998Crossref PubMed Scopus (10) Google Scholar). Here, we have mapped the YopO phosphorylation sites on the substrate gelsolin via proteomic analysis and have determined the effects of phosphorylation on the activities of gelsolin in actin filament dynamics. We identified Ser-385 as the major YopO phosphorylation site on gelsolin. YopO-phosphorylated gelsolin has actin-severing activity in the presence of EGTA. To validate the effect of phosphorylation at these sites, phosphomimetic mutants were created to mimic the phosphorylated state of gelsolin. Although WT gelsolin remains inactive in calcium-free conditions, the phosphomimetic mutants exhibit varying degrees of activity in depolymerization and severing actin filaments in both sedimentation and pyrene-actin assays. Mutants incorporating both S381D and S385D, PM5 (S381D/S384D/S385D), followed by PM1 (S381D/S385D), display the strongest activity in calcium-free conditions. However, single phosphomimetic mutants did not display any activity in EGTA conditions, probably due to the inability of substitution by aspartic acid to completely mimic phosphorylation. Nevertheless, phosphomimetic mutants PM1 (S381D/S385D), PM2 (S381D), PM3 (S385D), and PM5 (S381D/S384D/S385D) exhibit reduction in nucleation activity. Actin nucleation occurs through binding to two actin monomers in an appropriate orientation (26Hesterkamp T. Weeds A.G. Mannherz H.G. The actin monomers in the ternary gelsolin: 2 actin complex are in an antiparallel orientation.Eur. J. Biochem. 1993; 218: 507-513Crossref PubMed Scopus (65) Google Scholar), which in the case of gelsolin involves the linker between G3 and G4 wrapping around the filament (8Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. The crystal structure of plasma gelsolin: implications for actin severing, capping, and nucleation.Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The reduction in nucleation by phosphorylated gelsolin may be due to the interference of the phosphorylated sites in adopting a conformation that is optimal for actin nucleation. However, inhibiting nucleation by gelsolin is unlikely to have a physiological role in vivo, because pointed-end elongation is not favored in the presence of profilin in vivo. The identification of phosphorylation sites within the linker region of gelsolin and its effects on gelsolin activity support a role for the linker in regulating the activity of gelsolin. YopO phosphorylation sites Ser" @default.
• W2593236198 created "2017-03-16" @default.
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• W2593236198 modified "2023-10-13" @default.
• W2593236198 title "Yersinia effector protein (YopO)-mediated phosphorylation of host gelsolin causes calcium-independent activation leading to disruption of actin dynamics" @default.
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