Purification of Erythrocyte Dematin (protein 4.9) Reveals an Endogenous Protein Kinase That Modulates Actin-bundling Activity

Husain-Chishti, A.; Faquin, William C.; Wu, Çherry; Branton, Daniel

doi:10.1016/s0021-9258(18)81891-9

Cited by 48 publications

(4 citation statements)

References 34 publications

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“…The concentration of transgelin (an average of 2.4/zM) required to gel 9.3/~M actin is slightly more than the apparent saturation concentration (1.5/~M) which we found in our experiments. While this may well be due to actin induced self association of transgelin, the level of saturation is less than that of an actin bundling protein found in L/mulus (46) and similar to dematin (12). The interaction of transgelin with skeletal muscle actin is not surprising, even although it is clearly absent from this tissue in vivo (41), given the high degree of conservation between actin isoforms in different tissues and widely divergent species (15,34,35,44,56).…”

Section: Discussionmentioning

confidence: 91%

Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.

Shapland

Hsuan

Totty

et al. 1993

The Journal of Cell Biology

156

152

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Abstract. We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P. Lowings, and D. Lawson. 1988. J. Cell Biol. 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast. The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of •7.5 x 105 M-~; and (b) causes actin filament gelation within 2 min. Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions. EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min. By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed. Since the protein is transformation sensitive and gels actin, we have named it transgelin. TIN is crucial for a variety of cellular events such as motility, division and cell surface receptor movement (40,43,55,56). Actin organization can be controlled by a large number ('~70 are so far known) (3, 37) of actin-associated proteins which act by bundling, cross-linking, severing, gelating, sequestering monomers, or preventing actin polymerization (3,9,37,40,43,44,49). These molecules, acting either individually or in concert, regulate (a) the physical status of actin (that is the ratio of globular to filamentous actin) (43, 56), (b) actin geometry (5,19,20,27,44), and (c) provide both the fine control and the driving force required for the cellular events mentioned above.Disruption of the actin network is known to accompany events such as neoplasia (21, and see reference 4D and, in this instance, can involve alterations to both the actin microfilament network itself (21) and the expression of selected actin-associated proteins (45). However, only seven of the 70 or so proteins associated with actin are thus far known to be affected by transformation; namely, the higher molecular weight tropomyosins (11), nonmuscle caldesmon (14), smooth muscle myosin light chain 2 (16), gelsolin and actin-binding protein (17), protein C4 (41), and gelsolin (50). Since some actin-associated proteins probably act synergistically to control and organize actin, e.g., tropomyosin, gelsolin, and caldesmon (50), it seems very likely that the major changes to the actin microfiament network that occur following transformation may reflect the coordinated downregulation of several of these important molecules rather than alterations to the ratio of globular: filamentous actin itself.We have previously identified a transformation-sensitive polypeptide doublet (protein C4h'9 present in all cells and tissues apart from skeletal muscle, red blood ceils and neurons, and have shown that the higher rela...

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Section: Discussionmentioning

confidence: 91%

Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.

Shapland

Hsuan

Totty

et al. 1993

The Journal of Cell Biology

156

152

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“…6 The homologous headpiece is also present at the C-termini of certain proteins that lack the gelsolin core, as exemplified by dematin found in red blood cells of vertebrates. 7 In vertebrate villins, the C-terminal headpiece domain is linked to the N-terminal gelsolin core through an unstructured, nonconserved 40-residue polypeptide (the "linker"), the longest interdomain linker in the protein. 8 Analogous core-toheadpiece linkers of highly varying length, amino-acid composition, and degree of sequence conservation are also present in most villin homologues (Supplementary Figure 1).…”

mentioning

confidence: 99%

“…The headpiece in turn consists of two distinct subdomains: a more thermodynamically stable C-terminal subdomain and less stable N-terminal one . The homologous headpiece is also present at the C-termini of certain proteins that lack the gelsolin core, as exemplified by dematin found in red blood cells of vertebrates . In vertebrate villins, the C-terminal headpiece domain is linked to the N-terminal gelsolin core through an unstructured, nonconserved 40-residue polypeptide (the “linker”), the longest interdomain linker in the protein .…”

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confidence: 99%

Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin

et al. 2018

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Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences. It is unknown to what extent the plant villin headpiece structure and function resemble those of the well-studied vertebrate counterparts. Here we present the first solution NMR structure and backbone dynamics characterization of a headpiece from plants, villin isoform 4 from Arabidopsis thaliana. The villin 4 headpiece is a 63-residue domain (V4HP63) that adopts a typical headpiece fold with an aromatics core and a tryptophan-centered hydrophobic cap within its C-terminal subdomain. However, V4HP63 has a distinct N-terminal subdomain fold as well as a novel, high mobility loop due to the insertion of serine residue in the canonical sequence that follows the variable length loop in headpiece sequences. The domain binds actin filaments with micromolar affinity, like the vertebrate analogues. However, the V4HP63 surface charge pattern is novel and lacks certain features previously thought necessary for high-affinity F-actin binding. Utilizing the updated criteria for strong F-actin binding, we predict that the headpiece domains of all other villin isoforms in A. thaliana have high affinity for F-actin.

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“…By contrast, the binding of protein 4.1 to inside-out vesicles was not affected by phosphorylation by the cAMP-dependent protein kinase (Danilov et al, 1990). Recently, Husain-Chishti et al (1988, 1989 reported that the actin-bundling activity of protein 4.9 was abolished by phosphorylation by the cAMP-dependent protein kinase, but not by protein kinase C. Although spectrin constitutes the major protein of the cytoskeletal meshwork, the phosphorylation of spectrin, on the other hand, was found to be "silent" and exhibited no meaurable effect on its ability to interact with other cytoskeletal proteins (Lu et al, 1985;Eder et al, 1986). Taken together, these data suggest that phosphorylation may provide a mechanism for maintaining a relaxed, flexible cytoskeletal structure that is essential for the normal function of the red cell membrane.…”

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confidence: 99%

Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

Chao¹,

Tao²

1991

Biochemistry

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The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other hand, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)

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Purification of Erythrocyte Dematin (protein 4.9) Reveals an Endogenous Protein Kinase That Modulates Actin-bundling Activity

Cited by 48 publications

References 34 publications

Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.

Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.

Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin

Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

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