Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments

Abstract

Dictyostelium actin was shown to become phosphorylated on Tyr-53 late in the developmental cycle and when cells in the amoeboid stage are subjected to stress but the phosphorylated actin had not been purified and characterized. We have separated phosphorylated and unphosphorylated actin and shown that Tyr-53 phosphorylation substantially reduces actin's ability to inactivate DNase I, increases actin's critical concentration, and greatly reduces its rate of polymerization. Tyr-53 phosphorylation substantially, if not completely, inhibits nucleation and elongation from the pointed end of actin filaments and reduces the rate of elongation from the barbed end. Negatively stained electron microscopic images of polymerized Tyr-53-phosphorylated actin show a variable mixture of small oligomers and filaments, which are converted to more typical, long filaments upon addition of myosin subfragment 1. Tyr-53-phosphorylated and unphosphorylated actin copolymerize in vitro, and phosphorylated and unphosphorylated actin colocalize in amoebae. Tyr-53 phosphorylation does not affect the ability of filamentous actin to activate myosin ATPase.

Fig. 1.

Conditions that increase pY-actin in Dictyostelium. (A) Relative amounts of pY-actin in vegetative (V) and starved (S) amoebae and fruiting bodies (F) by immunoblotting with anti-pY antibody. (B) Increase of pY-actin in cells exposed to CdCl₂, heat, NaN₃, or PAO. (C) Increase in pY-actin with days in culture. (D) Specificity of anti-phosphotyrosine antibody. Immunoblotting of phosphorylated actin is blocked by 1 mM pY but not by 1 mM pS or 1 mM pT. CB, Coomassie blue; pY, phosphotyrosine; pS, phosphoserine; pT, phosphothreonine.

Fig. 2.

Colocalization of unphosphorylated and pY-actin. (A) Localization of pY-actin in vegetative, polarized, and dividing cells. (B) Localization of total F-actin (rhodamine phalloidin, red) and pY-actin (anti-pY, green).

Fig. 3.

Large-scale purification of actin. (A) Two-dimensional gel of total cell lysate stained with Coomassie blue (CB), and anti-actin and anti-pY antibodies. (B) CB-stained gel of SDS/PAGE of purified actin before separation of phosphorylated and unphosphorylated actin. (C) Two-dimensional gel of sample in B stained with CB and anti-actin and anti-pY-antibodies.

Fig. 4.

Separation of pY-actin and unphosphorylated actin. (A) Elution profile of Mono P column of the actin sample shown in Fig. 3. (B) SDS/PAGE of fractions eluted between 60.5 and 70.5 ml stained with Coomassie blue (CB) and anti-actin and anti-pY antibodies. (C) Two-dimensional gel of pooled fractions of unphosphorylated actin (Actin) and P-actin and a 1:1 mixture of the purified proteins.

Fig. 5.

HPLC of LysC-digests of unphosphorylated actin (Actin) and pY-actin (P-Actin). A and B identify the only peptides that differ in the two samples.

Fig. 6.

Concentration dependence of inhibition of pancreatic DNase I by unphosphorylated actin and pY53-actin. (Inset) Amplification of the region between 0 and 4 nM unphosphorylated actin.

Fig. 7.

Critical concentrations and copolymerization of unphosphorylated actin and pY53-actin. (A) Critical concentration curves. Actin and pY53-actin at the indicated concentrations were incubated for 24 h at room temperature. (B) Unphosphorylated actin (8 μM), p53-actin (0.2 μM), or a mixture of unphosphorylated actin (7.8 μM) and pY53-actin (0.2 μM) were incubated at room temperature for 24 h and centrifuged at 240,000 × g for 1 h in a Beckman (Fullerton, CA) TL100 centrifuge. SDS/PAGE aliquots of the supernatants (S), pellets (P), and total sample before centrifugation (T) were stained with Coomassie blue and anti-actin and anti-pY-antibodies.

Fig. 8.

Polymerization kinetics and myosin ATPase activation of unphosphorylated actin and pY53-actin. (A) Four-micromolar pY53-actin was polymerized alone (control) or in the presence of 20 nM gelsolin or 30 nM Arp2/3-VCA. (B) Unphosphorylated actin (4 μM) polymerized alone or in the presence of 20 nM gelsolin or 30 nM Arp2/3-VCA. (C) Correlation between ATP hydrolysis (circles) and spectrin–actin- (1.3 nM) nucleated polymerization (triangles) of 7.6 μM unphosphorylated actin (filled symbols) and 7.6 μM pY53-actin (open symbols). (D) ATPase activity of 150 nM Dictyostelium myosin II S1 as a function of the concentration of unphosphorylated and pY53-phosphorylated actin.

Fig. 9.

Electron microscopic images of polymerized unphosphorylated and pY53-actin. (A) Filaments of unphosphorylated actin. (B) Short bars of polymerized pY53-actin. (C) Mixture of short bars and filaments of pY53-actin. (D) Same sample of pY53-actin as in C, but with the addition of myosin S1. Insets in A and B are enlarged 2-fold.