p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I - PubMed (original) (raw)

Comparative Study

p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I

H Brzeska et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg2+-ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P(i) per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.

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Figures

Figure 1

Figure 1

Substrate specificities of Acanthamoeba MIHCK, 35K, and PAK1. Kinase activities were determined as described with the synthetic peptides described in Table 1 as substrates. MIHCK was fully activated by autophosphorylation (11) before its assay; expressed 35K is fully active without autophosphorylation (17). His-PAK1 is constitutively active (U.G.K., unpublished observations). To compare their relative substrate specificities, the activities were normalized to their activities with PC9 as substrate as 100; the actual specific activities with PC9 were: MIHCK, 26 μmol·min−1·mg−1; 35K, 27 μmol·min−1·mg−1; and His-PAK1, 0.37 μmol·min−1·mg−1.

Figure 2

Figure 2

Autoradiography of the heavy chain of Acanthamoeba myosin IC after phosphorylation with expressed 35K and PAK1. Myosin IC was incubated with [γ-32P]ATP and either 35K (Left) or His-PAK1 (Right) as described for the indicated times. The reactions were stopped by addition of SDS sample buffer and the samples separated by PAGE. The autoradiogram of the myosin IC heavy chains is shown. The level of phosphorylation was ≈1 mol of Pi per mol in all samples except the 5-min 35K sample, which was ≈0.8 mol of Pi per mol.

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