In search of the regulation mechanisms for isoform specific myosin assembly, we have used the COOH-terminal fragments of nonmuscle myosin isoforms MIIA and MIIB (MIIA(F46) and MIIB(alpha)(F47)) as a model system. Phosphorylation by protein kinase C (PK C) or casein kinase II (CK II) within or near the nonhelical tail-end domain inhibits assembly of MIIB(alpha)(F47) [Murakami, N., et al. (1998) Biochemistry 37, 1989]. In the study presented here, we mutated the kinase sites to analyze the inhibition mechanisms of MIIB assembly by phosphorylation. Replacement of the CK II or PK C sites with Asp (MIIB(alpha)(F47)-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg(2+), by significantly increasing the critical concentrations for assembly. Without Mg(2+), MIIB(alpha)(F47)-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIB(alpha)(F47) by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg(2+), MIIB(alpha)(F47)-wt promoted assembly of MIIB(alpha)(F47)-CK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIB(alpha)(F47)-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIB(alpha)(F47)-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca(2+)-dependent manner to MIIA(F46), but not appreciably to MIIB(alpha)(F47). At 0.15 M NaCl, mts 1-Ca(2+) not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIB(alpha)(F47) in MIIA(F46) and MIIB(alpha)(F47) mixtures, indicating that mts 1 is an inhibitor specific to MIIA assembly. Our results suggest strongly that assembly of MIIA and MIIB is regulated by distinct mechanisms via tail-end domains: phosphorylation of MIIB and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.cell motility | lamellipodium | Rac | Arp2/3-complex | Hssh3bp1

The av/41 polymorphism of erythroid a-spectrin has been characterized initially by an increased susceptibility to proteolysis of the aIV-aV domain junction (Alloisio N

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Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of ␤-spectrin with a complementary region near the C-terminal of ␣-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of ␤-spectrin required for association with ␣-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent ␤-spectrin peptides was particularly critical. The beginning of the first homologous motif for both ␤-spectrin and the related dimerization site of ␣-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif ␤-spectrin peptide (␤1-4 ؉ ) with this earlier starting point bound to full-length ␣-spectrin with a K d of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N-and C-terminal truncations of one or more motifs from ␤1-4 ؉ showed that the first motif was essential for dimerization since its deletion abolished binding, but ␤1؉ alone could not associate with ␣-monomers. The first two motifs (␤1-2 ؉ ) represented the minimum lateral dimer assembly site with a K d of about 230 nM for interaction with full-length ␣-spectrin or an ␣-spectrin nucleation site recombinant peptide, ␣18 -21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nm, while each additional motif added only 0.85 nm to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.The membrane skeleton of the human erythrocyte consists of a network of proteins that associates with the inner surface of the cell membrane and imparts remarkable structural integrity and flexibility to circulating erythrocytes. Spectrin is the major structural component of this specialized submembranous protein network. The basic functional unit of spectrin is a heterodimer formed by side-to-side, antiparallel association of a 280-kDa ␣ subunit with a 246-kDa ␤ subunit. Spectrin dimers associate head-to-head to form tetramers, the predominant form of spectrin in the membrane skeleton. These tetramers cross-link short actin oligomers, an association modulated by band 4