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.

Fragmin, a microfilament regulatory protein from Physarum polycephalum, is phosphorylated by casein kinase II-type enzymes.

Fragmin is a 42 kDa regulatory protein involved in actin microfilament organization in Physarum polycephalum. We show that fragmin is a target of casein kinase II (CK II) enzymes isolated from evolutionarily divergent species. In Physarum microplasmodia, two such kinases were identified. A serine residue located in the sequence Gly-Gly-Ser-Asp-Leu-Glu constitutes the phosphorylation site and was identified by phosphopeptide sequencing, mass spectometry analysis, and inhibition studies with a synthetic peptide corresponding to this site. Interestingly, the actin-fragmin dimer (A--F) as well as the actin2-fragmin trimer (A2--F) are equally efficient targets, and phosphorylation had no effect on the actin-binding properties of fragmin. Actin-fragmin isolated from microplasmodia revealed a minor acidic fragmin isoform, suggesting that fragmin is phosphorylated in vivo. The actin-fragmin complex is also phosphorylated on the actin subunit by an endogenous actin-fragmin kinase [Gettemans, J., De Ville, Y., Vandekerckhove, J., & Waelkens, E. (1992) EMBO J. 11, 3185-3191]. We show that the two phosphorylation events act independently of each other.

The actin-binding properties of the Physarum actin-fragmin complex. Regulation by calcium, phospholipids, and phosphorylation.

The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with respect to regulation by Ca2+, phospholipids, and phosphorylation of the actin subunit by the endogenous actin-fragmin kinase. Fragmin possesses two high affinity actin-binding sites and probably also a third, low affinity site. Its nucleating and F-actin severing activities are inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin-fragmin specifically binds PIP2 which competes with actin for the Ca(2+)-sensitive site. However, PIP2 cannot dissociate the actin-fragmin complex nor the actin2-fragmin trimer. Efficient F-actin nucleating activity by actin-fragmin is only observed with unphosphorylated actin-fragmin, in the absence of PIP2 and at high Ca2+ (> microM) concentrations. In the presence of PIP2, actin-fragmin only caps actin filaments when unphosphorylated. The results suggest that in the cell, hydrolysis of PIP2, concomitant with the increase of cytosolic Ca2+, could promote subcortical actin polymerization.

Microfilament dynamics: regulation of actin polymerization by actin-fragmin kinase and phosphatases.

Based on the phosphorylation of the purified actin-fragmin complex, an 80 kDa monomeric kinase (AFK) has been isolated from Physarum polycephalum. Protein chemical analysis and studies involving kinase inhibitors and effectors establish that the AFK is a unique kinase that cannot be classified so far in one of the conventional kinase families. The actin-fragmin kinase behaves as an "independent" kinase since its activity towards the actin-fragmin complex is apparently not regulated by the binding of a ligand (e.g., the cyclic-nucleotides, Ca2+, calmodulin, phosphatidylserine and diolein). Rigorous screening of the substrate specificity suggests that the actin-fragmin complex represents the only substrate for this kinase. This kinase phosphorylates the actin moiety of the actin-fragmin complex at two consecutive threonine residues which constitute one of the contact sites for DNase I (37) and which are also located at one of the proposed actin-actin contact sites along the long-pitch helix of F-actin (38, 39). The physiological importance of this phosphorylation was demonstrated by studying the effect of phosphorylation on the nucleation and the capping activity of the actin-fragmin complex using fluorescence enhancement analysis. As could be demonstrated, the nucleation of actin filaments by the actin-fragmin complex is completely abolished upon phosphorylation by the AFK. Phosphorylation of the complex also interferes with its capping activity, which becomes Ca(2+)-dependent. In addition, capping and nucleating activity is regulated in vitro by phosphoinositides, of which PIP2 displays the highest activity and specificity. PIP2 partially inhibits the nucleation and capping activity of the unphosphorylated actin-fragmin. The capping activity of the phosphorylated actin-fragmin complex was inhibited by PIP2 to a much greater extent as compared to the unphosphorylated actin-fragmin complex. Among all phospholipids tested, PIP2 displayed the highest specificity. Initial experiments with purified preparations of the PP-1, PP-2A, PP-2B, alkaline phosphatase and acid phosphatases showed that PP-1 and PP-2A phosphatases were capable of dephosphorylating the phospho actin-fragmin complex. These findings raised the question of whether these or other protein phosphatases were involved in the dephosphorylation of this substrate in vivo. To address this question, Physarum extracts were subjected to fractionation by ion exchange chromatography, and the column fractions were assayed in a variety of conditions, to identify the protein phosphatases involved in the dephosphorylation of this substrate and to identify the elution position of the major Ser/Thr protein phosphatases present in the Physarum extract.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and partial amino acid sequence of the actin-fragmin kinase from Physarum polycephalum.

The 80-kDa actin-fragmin kinase (AFK) was purified from Physarum polycephalum microplasmodia to apparent homogeneity through a procedure involving six chromatographic steps. Taking the activity at the first purification step as 100%, the kinase was purified more than 1500-fold, with an overall yield of 8%. The specific activity of the purified enzyme was 700 U/mg. The total amount of AFK present could be estimated as 34 ng/mg extracted protein. One of the polyclonal antibodies raised against four tryptic peptides of the purified 80-kDa AFK recognized the 80-kDa band in an immunoblot and could inhibit the AFK activity up to 100%. A minor 78-kDa protein, also displaying AFK activity, appeared to be derived from the 80-kDa AFK. A partial amino acid sequence analysis, covering up to 20% of the protein (150 amino acids), was performed and confirmed the lack of similarity with any of the sequenced kinases. These data are in agreement with the unique physical and enzymatic properties of the AFK.

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].