L-Plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages

Podosomes are cellular structures acting as degradation ‘hot-spots’ in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages

Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase.

The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49]

A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.

Actin-fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues. Whereas the N-terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full-length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N-terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P-loop). However, introduction of two different point mutations into this putative P-loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non-muscle cells

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid.

An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides

Identification of Tyr438 as the major in vitro c-Src phosphorylation site in human gelsolin: a mass spectrometric approach.

Gelsolin is an actin-binding protein (82 kDa) consisting of six repeated segments (S1-S6), each approximately 120 residues long. It interacts with phospholipids and we previously showed that phosphatidylinositol 4,5-bisphosphate promotes phosphorylation of gelsolin by the tyrosine kinase c-Src. We used a combination of different methods, such as thin-layer chromatography and anti-phosphotyrosine-agarose immunoprecipitation of phosphopeptides combined with matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and post source decay (PSD) analysis, to identify the phosphorylation sites in gelsolin. The major phosphorylation site (Tyr438) was located in subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin-actin2 complex was inhibited by 90%. Gelsolin phosphorylation by c-Src in the presence of lysophosphatidic acid also revealed Tyr438 as the most prominent site. Additional minor sites were found using the anti-phosphotyrosine bead immunoprecipitation method followed by MALDI-MS and PSD analysis. These sites, representing approximately 5% of the total phosphate incorporation, were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we generated antibodies which specifically recognize Tyr438 phosphorylated gelsolin

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.

Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity

The role of gelsolin domain 3 in familial amyloidosis (Finnish type)

In the disease familial amyloidosis, Finnish type (FAF), also known as AGel amyloidosis (AGel), the mechanism by which point mutations in the calcium-regulated actin-severing protein gelsolin lead to furin cleavage is not understood in the intact protein. Here, we provide a structural and biochemical characterization of the FAF variants. X-ray crystallography structures of the FAF mutant gelsolins demonstrate that the mutations do not significantly disrupt the calcium-free conformations of gelsolin. Small-angle X-ray–scattering (SAXS) studies indicate that the FAF calcium-binding site mutants are slower to activate, whereas G167R is as efficient as the wild type. Actin-regulating studies of the gelsolins at the furin cleavage pH (6.5) show that the mutant gelsolins are functional, suggesting that they also adopt relatively normal active conformations. Deletion of gelsolin domains leads to sensitization to furin cleavage, and nanobody-binding protects against furin cleavage. These data indicate instability in the second domain of gelsolin (G2), since loss or gain of G2-stabilizing interactions impacts the efficiency of cleavage by furin. To demonstrate this principle, we engineered non-FAF mutations in G3 that disrupt the G2-G3 interface in the calcium-activated structure. These mutants led to increased furin cleavage. We carried out molecular dynamics (MD) simulations on the FAF and non-FAF mutant G2-G3 fragments of gelsolin. All mutants showed an increase in the distance between the center of masses of the 2 domains (G2 and G3). Since G3 covers the furin cleavage site on G2 in calcium-activated gelsolin, this suggests that destabilization of this interface is a critical step in cleavage