Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring.

Abstract

Abstract. nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including t~-actinin,/3-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each ~96 residues long, predicted to have stretches of/3-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotaryshadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin illaments. The large size of the leaves, their interruption by two hinges and flexible actin-binding site, facilitate cross-linking of widely dispersed actin filaments.N ONMUSCLE cells achieve a wide variety of shapes by arranging peripheral linear actin filaments in different ways. Actin-modulating proteins are responsible for this versatility by adjusting actin filament length and by defining the organization of actin filaments and their binding to other cellular components (54, 56). Actin-binding protein (ABE also called nonmuscle filamin) ~ was among the first such proteins discovered (25,55,62) and has a broad phylogenetic distribution (3,4,34,47,49,51,59,60,63). ABP from most cell types examined promotes high-angle

Rho-kinase contributes to diphosphorylation of myosin II regulatory light chain in nonmuscle cells

“…The numbers on panel show fraction numbers. Asterisk and closed arrowhead indicate the components of 250 kDa (p250) and 160 kDa (p160), respectively (ABP-280) (Gorlin et al, 1990). These results showed that Rho-kinase has an activity for MRLC diphosphorylation in nonmuscle cells.…”

Filamin-regulated F-actin Assembly Is Essential for Morphogenesis and Controls Phototaxis in Dictyostelium

“…Structurally filamins are homodimers with large polypeptide chains that associate at their C termini (24,25). Their conserved N-terminal actin-binding domains (ABD) 2 belong to the ABDs common to the members of the ␣-actinin/spectrin superfamily of actin-binding proteins (26,27).…”

Filamin structure, function and mechanics: are altered filamin-mediated force responses associated with human disease?

“…FLNA is a substrate for multiple kinases (reviewed in Sarkisian et al 2008) depending on whether or not its actin bound (Kawamoto and Hidaka 1984), with its cross-linking activity itself regulated by phosphorylation (Cukier et al 2007). FLN is a substrate for the Ca 2+ -activated protease calpain (Gorlin et al 1990) and for the ASB2 ubiquitin ligase (Heuze et al 2008) which provide irreversible regulatory mechanisms for severing FLN cross-links in processes requiring membrane and cytoskeleton remodelling such as cell migration (Marzia et al 2006;Baldassarre et al 2009) and receptor cycling (Seck et al 2003). FLN's role in cell motility and spreading appears to be cell-type dependent.…”

“…The hypothesis that FLN acts as a molecular spring was initially formulated upon sequence analysis of FLN domain structure (Gorlin et al 1990) with subsequent analysis of its biomechanical and structural properties showing it suited to a cellular role regulating both the organization and mechanics of F-actin networks. FLN is found in the F-actin rich region near cell membranes where fast remodelling of the cytoskeleton is required, e.g.…”

“…FLNA and FLNB respond similarly during in vitro rheological measurements showing resistance to 30-and 10-Pa shear stress, respectively, but in contrast, FLNA/B isoforms missing the first hinge region showed decreased elasticity in response to applied stress breaking at lower applied stress, 0.1 Pa, even though the cross-linking gelation concentration was equivalent to full-length FLN (Gardel et al 2006). The absence of hinge I also affects regulation of cross-linking by proteolysis since this hinge contains the calpain cleavage site (Gorlin et al 1990).…”