The sarcolemma of fast-twitch skeletal muscle fibers is organized into costameres that are linked to the Z-disks and M-lines of the underlying contractile apparatus, and, in some fibers, are also present in longitudinally oriented structures. Because the disruption of costameres may contribute to the instability of the sarcolemma observed in various myopathies and muscular dystrophies, we have been studying their organization and the biochemical basis for their formation. Previous results suggest that both actin and desmin are involved in linking the contractile apparatus to the sarcolemma (Rybakova et al., 2000
; O'Neill et al., 2002
), but because these proteins are concentrated only at the level of Z-disks, they are unlikely to be able to link the M-line and longitudinally oriented domains of costameres to the contractile apparatus. We have cloned K8 and K19 from striated muscle and shown them to be present at all three domains of costameres (Ursitti et al., 2004
). We postulated that the filaments formed by K8 and K19 could interact directly with the N-terminal ABD of dystrophin, much as neurofilaments, and vimentin and plectin, associate with the ABDs of βII-spectrin and fimbrin, respectively (Correia et al., 1999
; Macioce et al., 1999
; Sevcik et al., 2004
). Here, we confirm this prediction, characterize the specificity of the association, and demonstrate its potential importance in situ.
Many of our experiments use transfection of COS-7 cells to examine interactions between cytokeratins and ABDs under conditions prevalent in mammalian cytoplasm. Although COS cells fail to show a clear association of full-length dystrophin with actin filaments (Lee et al., 1991
), they have proved useful for our studies because the distribution of ABDs, and especially the Dys-ABD (expressed without the rod and C-terminal regions), can be easily localized to actin or other filamentous structures. Untransfected COS-7 cells have intermediate filaments, of course, but unlike K8/K19 filaments, these do not associate avidly with the Dys-ABD, although they do so occasionally (Ursitti et al., 2004
), perhaps due to the presence of low levels of K19 or other subunits to which it can bind (see below). More frequently, the Dys-ABD codistributes with actin in stress fibers and at the cell periphery. Both the endogenous intermediate filaments and actin-rich structures are easily distinguished from the structures formed after overexpression of cytokeratins. This facilitated the discovery of how efficiently the Dys-ABD colocalized with filaments formed by the overexpression of K8 and K19.
The association of the Dys-ABD with actin in endogenous stress fibers and at the plasma membrane of most COS-7 cells is consistent with in vitro studies of its ability to bind to actin and to cross-link microfilaments (Senter et al., 1993
; Renley et al., 1998
; Sutherland-Smith et al., 2003
). Despite its cross-linking activity, linked to its ability to dimerize (Sutherland-Smith et al., 2003
), the Dys-ABD does not significantly alter the organization of K8/K19 filaments, suggesting that the Dys-ABD does not cross-link these filaments to each other. It also seems unable to cross-link K8/K19 filaments to actin (), suggesting that it is incapable of binding to both types of filamentous proteins simultaneously. Instead, it associates preferentially with K19, without changing the distribution of the aggregates or filaments formed by this cytokeratin.
The specificity of the Dys-ABD for K19 and the filaments it forms with K8 is remarkable, indeed. The Dys-ABD does not associate to a significant extent with filaments composed of desmin or of K8 paired with K18 (like K19, a type I cytokeratin), nor does the homologous ABD of βI-spectrin associate with K8/K19 filaments. The ability of the Dys-ABD to bind purified K19 in vitro indicates that binding is direct as well as specific, but we cannot rule out the possibility that other endogenous proteins promote their association in vivo. Although, in addition to K8, K19 also can copolymerize with K5 and K7 (Coulombe and Omary, 2002
), we are not aware of any evidence of K5 or K7 in striated muscle and have so far been unsuccessful in detecting transcripts encoding K5 in that tissue (Ursitti, Lee, McNally, and Bloch, unpublished data). More extensive studies of the cytokeratins and their roles in muscle are clearly needed.
The basis for the specificity of binding of the Dys-ABD to K19 remains unclear. One possibility is that the Dys-ABD shares some homology with sequences within the cytokeratins that are required for oligomerization. We found significant homology between amino acids 42-65 of the Dys-ABD and two distinct sequences shared by K19 (amino acids 180-209 and 294-320), and by K8 (amino acids 190-219 and 305-332) (). Although these sequences share other key features, we suggest the term “I/LE(G)L motif “ for this region, for the amino acid residues that are conserved in four of the five sequences (; the G is in parentheses to indicate that it is missing in one sequence). Although not unique to cytokeratins 8 and 19, these amino acids are not conserved in human K18, with which K8 can heterodimerize, or in human K5 or K7, with which K19 can heterodimerize. It is also absent in other classes of intermediate filament proteins, including type III proteins (desmin, vimentin, synemin, or GFAP), neurofilaments (L, M, or H) and nuclear lamins, consistent with the specificity we have described for the interaction of Dys-ABD with K8/K19 filaments. Notably, the I/LE(G)L motif is present only in the first, more N-terminal CH domain of the Dys-ABD. Preliminary experiments confirm that this domain, and not the more COOH-terminal CH domain of the Dys-ABD, has binding activity for K19, in agreement with a recent report that the first, more N-terminal CH domain of plectin harbors the binding activity of the plectin ABD for vimentin (Sevcik et al., 2004
Figure 9. Sequence homologies between the Dys-ABD and cytokeratins 8 and 19. The sequence of the C-helix and nearby residues of the Dys-ABD (human, accession number NP031894) was compared with the sequences of K8 (human, accession number P05787) and K19 (human, (more ...)
Structural studies show that the sequences containing the I/LE(G)L motifs of the Dys-ABD, K8, and K19 are all helical, although the flanking N- and C-terminal residues in the Dys-ABD are in nonhelical linkers (Norwood et al., 2000
). The I/LE(G)L motif of the Dys-ABD, contained in its third or C-helix, has not been implicated in actin binding (Norwood et al., 2000
; Gimona et al., 2002
), nor is it present in human actins. It contains two residues, Q45 and D52, that distinguish it from the homologous region in the ABD of βI-spectrin and that may account in part for its preference for the cytokeratins. The Dys-ABD shares D52, but not Q45, with the ABD of utrophin, suggesting that this residue may contribute to cytokeratin binding. Both these side chains are exposed to the solvent in a shallow groove on the surface of the Dys-ABD (Norwood et al., 2000
) and so could help form a binding site. Site-directed mutagenesis will be required to test this idea further.
Whatever confers the specificity of the Dys-ABD for K19, our results suggest that the binding is of only moderate affinity. Values in the low micromolar range are consistent with the rapid on- and off-rates for binding observed in surface plasmon resonance experiments at the micromolar concentrations of reagents we used () as well as with the results of our dot blots. This range is comparable to the affinity of the Dys-ABD for actin filaments of ~10-50 μM (Senter et al., 1993
; Winder et al., 1995
; Renley et al., 1998
; Sutherland-Smith et al., 2003
). The affinity of intact, full-length dystrophin for actin is considerably higher, due to the presence of clusters of basic residues in its spectrin repeat region that greatly enhance actin binding (Amann et al., 1998
; Warner et al., 2002
). We have not yet assayed the other regions of dystrophin for their possible contributions to binding to cytokeratin filaments, nor have we examined any of the proteins that are normally complexed with dystrophin (Ahn and Kunkel, 1993
; Matsumura and Campbell, 1994
; Ozawa et al., 1998
) for similar activity. Nevertheless, the comparable affinities of the Dys-ABD for cytokeratin and actin filaments suggest that the severity of dystrophinopathies linked to mutations in the ABD may be due to changes in binding not only to actin but also to cytokeratins. This may explain the ability of a mutation (L54R) in the C-helix of the first CH domain of the Dys-ABD to cause Duchenne Muscular Dystrophy (Prior et al., 1993
The similarities and differences between the effects of overexpressing K8 and K19 alone in COS-7 cells and in muscle fibers are significant and revealing. In both types of cells, K8 incorporates into endogenous structures, whereas K19 forms intracellular aggregates. K19 in both cell types can associate with dystrophin (myofibers) or its ABD (COS-7), whereas K8 is targeted to other structures. This suggests that the COS-7 cell is a valid model in which to study some aspects of the behavior of cytokeratins of muscle. Nevertheless, the differences in the behavior of K8 and K19 in myofibers are considerable and potentially relevant to understanding the architecture of striated muscle cells in health and disease.
Unlike its activity in COS-7 cells, K8 overexpressed in skeletal myofibers seems to be unable to incorporate into endogenous cytokeratin filaments, which in the interior of striated muscle fibers are concentrated around Z-disks (Ursitti et al., 2004
). The fact that exogenous K8 concentrates midway between Z-disks suggests that A-bands or M-lines may harbor binding sites for this cytokeratin. Although this would be consistent with the presence of K8 at the domains of costameres that are linked to M-lines in underlying myofibrils, it does not explain its absence from the keratin filaments surrounding Z-disks. Access to those sites may be limited, perhaps by high local protein densities or by slow turnover of endogenous structures. More insight into these questions should be obtained through the identification of the proteins, other than the type I cytokeratin subunits, with which K8 interacts in striated muscle.
When expressed at high levels in myofibers, K19 disrupts costameres. Although we cannot rule out other plausible explanations for its activity, the ability of K19 to displace dystrophin from the sarcolemma in vivo is consistent with our studies of the association of the Dys-ABD to K19 in COS-7 cells. Whatever the mechanism, our results suggest that K19 can compete with the normal binding sites for dystrophin, which would otherwise concentrate at the sarcolemma. Because previous studies have shown that the costameric distribution of dystrophin-associated proteins, including β-dystroglycan, is compromised when dystrophin is absent from the sarcolemma in dystrophic muscle (Williams and Bloch, 1999b
), it is not surprising that the ability of β-dystroglycan to concentrate at costameres is altered in myofibers that overexpress K19. Dystroglycan also redistributes in the plane of the sarcolemma in other muscular dystrophies and myopathies (O'Neill et al., 2002
; Reed et al., 2004
Our finding that β-dystroglycan is absent from the vesicular structures that occur in many of the fibers that overexpress K19 is unexpected, however. It suggests that these vesicles, present in a subset of fibers that overexpress K19, represent a distinct membrane population that associates with membrane-cytoskeletal proteins, such as β-spectrin and dystrophin, but that does not contain all of the integral membrane proteins that anchor these proteins at the sarcolemma. The presence of β-spectrin at these structures is unlikely to be due to interactions of that molecule or its ABD with K19 directly (), but is instead more likely linked to the ability of β-spectrin to associate with muscle membranes in a manner that is independent of dystrophin (Porter et al., 1992
; Ehmer et al., 1997
; Williams and Bloch, 1999b
). Further research will be needed to establish the nature of the aggregates and vesicles induced by the overexpression of K19, the mechanism by which they form, and their relationship to structures seen in myopathies (De Bleecker et al., 1993
The ability of excess K19 to displace membrane-cytoskeletal proteins from the sarcolemma, to disrupt costameres, and to induce cytoplasmic aggregates and vesicles, reminiscent of vesicular myopathies (De Bleecker et al., 1993
), raises the possibility that some muscular dystrophies or myopathies may be caused not only by changes in the binding of the cytokeratins to the Dys-ABD but also by changes in their levels of expression, or by mutations that affect their ability to bind to each other. Studies of skeletal and cardiac muscle of mice lacking K8 or K19, due to homologous recombination, are now in progress in our laboratory to test this idea.