Myosin IIIB Uses an Actin-Binding Motif in Its Espin-1 Cargo to Reach the Tips of Actin Protrusions

We report a novel form of modulation of T-type calcium currents carried out by the neuronal actin-binding protein (ABP) Kelch-like 1 (KLHL1). KLHL1 is a constitutive neuronal ABP localized to the soma and dendritic arbors; its genetic elimination in Purkinje neurons leads to dendritic atrophy and motor insufficiency. KLHL1 participates in neurite outgrowth and upregulates voltage-gated P/Q-type calcium channel function; here we investigated KLHL1's role as a modulator of low-voltage-gated calcium channels and determined the molecular mechanism of this modulation with electrophysiology and biochemistry. Coexpression of KLHL1 with CaV3.1 or CaV3.2 (α1G or α1H subunits) caused increases in T-type current density (35%) and calcium influx (75–83%) when carried out by α1H but not by α1G. The association between KLHL1 and α1H was determined by immunoprecipitation and immunolocalization in brain membrane fractions and in vitro in HEK-293 cells. Noise analysis showed that neither α1H single-channel conductance nor open probability was altered by KLHL1, yet a significant increase in channel number was detected and further corroborated by Western blot analysis. KLHL1 also induced an increase in α1H current deactivation time (τdeactivation). Interestingly, the majority of KLHL1's effects were eliminated when the actin-binding motif (kelch) was removed, with the exception of the calcium influx increase during action potentials, indicating that KLHL1 interacts with α1H and actin and selectively regulates α1H function by increasing the number of α1H channels. This constitutes a novel regulatory mechanism of T-type calcium currents and supports the role of KLHL1 in the modulation of cellular excitability.

Download Full-text

Connecdenn 3/DENND1C binds actin linking Rab35 activation to the actin cytoskeleton

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0474 ◽

2012 ◽

Vol 23 (1) ◽

pp. 163-175 ◽

Cited By ~ 25

Author(s):

Andrea L. Marat ◽

Maria S. Ioannou ◽

Peter S. McPherson

Keyword(s):

Actin Cytoskeleton ◽

Membrane Trafficking ◽

Small Gtpase ◽

Actin Binding ◽

Binding Motif ◽

Endosomal Trafficking ◽

Nucleotide Exchange ◽

Endosomal Membrane ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors

The small GTPase Rab35 regulates endosomal membrane trafficking but also recruits effectors that modulate actin assembly and organization. Differentially expressed in normal and neoplastic cells (DENN)–domain proteins are a newly identified class of Rab guanine-nucleotide exchange factors (GEFs) that are grouped into eight families, each activating a common Rab. The members of one family, connecdenn 1–3/DENND1A–C, are all GEFs for Rab35. Why Rab35 requires multiple GEFs is unknown. We demonstrate that connecdenn 3 uses a unique C-terminal motif, a feature not found in connecdenn 1 or 2, to directly bind actin. This interaction couples Rab35 activation to the actin cytoskeleton, resulting in dramatic changes in cell shape, notably the formation of protrusive membrane extensions. These alterations are specific to Rab35 activated by connecdenn 3 and require both the actin-binding motif and N-terminal DENN domain, which harbors the GEF activity. It was previously demonstrated that activated Rab35 recruits the actin-bundling protein fascin to actin, but the relevant GEF for this activity was unknown. We demonstrate that connecdenn 3 and Rab35 colocalize with fascin and actin filaments, suggesting that connecdenn 3 is the relevant GEF. Thus, whereas connecdenn 1 and 2 activate Rab35 for endosomal trafficking, connecdenn 3 uniquely activates Rab35 for its role in actin regulation.

Download Full-text

Actin-binding motif of β-thymosins

Peptides 1994 ◽

10.1007/978-94-011-1468-4_273 ◽

1995 ◽

pp. 601-602

Author(s):

S. Stoeva ◽

G. Grübler ◽

W. Voelter

Keyword(s):

Actin Binding ◽

Binding Motif

Download Full-text

Primary structure and domain organization of human alpha and beta adducin.

The Journal of Cell Biology ◽

10.1083/jcb.115.3.665 ◽

1991 ◽

Vol 115 (3) ◽

pp. 665-675 ◽

Cited By ~ 103

Author(s):

R Joshi ◽

D M Gilligan ◽

E Otto ◽

T McLaughlin ◽

V Bennett

Keyword(s):

Amino Acids ◽

Sequence Similarity ◽

Complete Sequence ◽

Amino Acid Sequences ◽

Actin Binding ◽

Cell Contact ◽

Binding Motif ◽

Rat Tissues ◽

Erythroleukemia Cells ◽

Skeletal Protein

Adducin is a membrane-skeletal protein which is a candidate to promote assembly of a spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. The complete sequence of both subunits of human adducin, alpha (737 amino acids), and beta (726 amino acids) has been deduced by analysis of the cDNAs. The two subunits have strikingly conserved amino acid sequences with 49% identity and 66% similarity, suggesting evolution by gene duplication. Each adducin subunit has three distinct domains: a 39-kD NH2-terminal globular protease-resistant domain, connected by a 9-kD domain to a 33-kD COOH-terminal protease-sensitive tail comprised almost entirely of hydrophilic amino acids. The tail is responsible for the high frictional ratio of adducin noted previously, and was visualized by EM. The head domains of both adducin subunits exhibit a limited sequence similarity with the NH2-terminal actin-binding motif present in members of the spectrin superfamily and actin gelation proteins. The COOH-termini of both subunits contain an identical, highly basic stretch of 22 amino acids with sequence similarity to the MARCKS protein. Predicted sites of phosphorylation by protein kinase C include the COOH-terminus and sites at the junction of the head and tail. Northern blot analysis of mRNA from rat tissues, K562 erythroleukemia cells and reticulocytes has shown that alpha adducin is expressed in all the tissues tested as a single message size of 4 kb. In contrast, beta adducin shows tissue specific variability in size of mRNA and level of expression. A striking divergence between alpha and beta mRNAs was noted in reticulocytes, where alpha adducin mRNA is present in at least 20-fold higher levels than that of beta adducin. The beta subunit thus is a candidate to perform a limiting role in assembly of functional adducin molecules.

Download Full-text

Critical Roles of Phosphorylation and Actin Binding Motifs, but Not the Central Proline-rich Region, for Ena/Vasodilator-stimulated Phosphoprotein (VASP) Function during Cell Migration

Molecular Biology of the Cell ◽

10.1091/mbc.e01-10-0102 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2533-2546 ◽

Cited By ~ 89

Author(s):

Joseph J. Loureiro ◽

Douglas A. Rubinson ◽

James E. Bear ◽

Gretchen A. Baltus ◽

Adam V. Kwiatkowski ◽

...

Keyword(s):

Cell Motility ◽

Cell Movement ◽

Protein Family ◽

Actin Dynamics ◽

Actin Binding ◽

Binding Motif ◽

Conserved Sequence ◽

Cellular Processes ◽

Sequence Elements

The Ena/vasodilator-stimulated phosphoprotein (VASP) protein family is implicated in the regulation of a number of actin-based cellular processes, including lamellipodial protrusion necessary for whole cell translocation. A growing body of evidence derived largely from in vitro biochemical experiments using purified proteins, cell-free extracts, and pathogen motility has begun to suggest various mechanistic roles for Ena/VASP proteins in the control of actin dynamics. Using complementation of phenotypes in Ena/VASP-deficient cells and overexpression in normal fibroblasts, we have assayed the function of a panel of mutants in one member of this family, Mena, by mutating highly conserved sequence elements found in this protein family. Surprisingly, deletion of sites required for binding of the actin monomer-binding protein profilin, a known ligand of Ena/VASP proteins, has no effect on the ability of Mena to regulate random cell motility. Our analysis revealed two features essential for Ena/VASP function in cell movement, cyclic nucleotide-dependent kinase phosphorylation sites and an F-actin binding motif. Interestingly, expression of the C-terminal EVH2 domain alone is sufficient to complement loss of Ena/VASP function in random cell motility.

Download Full-text

Xin repeats define a novel actin-binding motif

Journal of Cell Science ◽

10.1242/jcs.01406 ◽

2004 ◽

Vol 117 (22) ◽

pp. 5257-5268 ◽

Cited By ~ 56

Author(s):

D. Pacholsky

Keyword(s):

Actin Binding ◽

Binding Motif

Download Full-text

Cancer-associated mutations in the protrusion-targeting region of p190RhoGAP impact tumor cell migration

The Journal of Cell Biology ◽

10.1083/jcb.201601063 ◽

2016 ◽

Vol 214 (7) ◽

pp. 859-873 ◽

Cited By ~ 15

Author(s):

Fabien Binamé ◽

Aurélien Bidaud-Meynard ◽

Laure Magnan ◽

Léo Piquet ◽

Bertille Montibus ◽

...

Keyword(s):

Cell Migration ◽

Tumor Cell ◽

Leading Edge ◽

Negative Regulator ◽

Actin Binding ◽

Tumor Cell Migration ◽

Spatiotemporal Regulation ◽

Localization Sequence ◽

Actin Protrusions ◽

And Function

Spatiotemporal regulation of RhoGTPases such as RhoA is required at the cell leading edge to achieve cell migration. p190RhoGAP (p190A) is the main negative regulator of RhoA and localizes to membrane protrusions, where its GTPase-activating protein (GAP) activity is required for directional migration. In this study, we investigated the molecular processes responsible for p190A targeting to actin protrusions. By analyzing the subcellular localization of truncated versions of p190A in hepatocellular carcinoma cells, we identified a novel functional p190A domain: the protrusion localization sequence (PLS) necessary and sufficient for p190A targeting to leading edges. Interestingly, the PLS is also required for the negative regulation of p190A RhoGAP activity. Further, we show that the F-actin binding protein cortactin binds the PLS and is required for p190A targeting to protrusions. Lastly, we demonstrate that cancer-associated mutations in PLS affect p190A localization and function, as well as tumor cell migration. Altogether, our data unveil a new mechanism of regulation of p190A in migrating tumor cells.

Download Full-text