Willard A. Bernbaum Cystic Fibrosis Research Center, Department of Pediatrics, Rainbow Babies and Childrens Hospital, and Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio
Submitted 1 October 2004 ; accepted in final form 7 December 2004
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ABSTRACT |
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truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter
We also discovered (15) colocalization of PKC- with F-actin in a Calu-3 airway epithelial cell line and avid binding of recombinant PKC-
to purified nonmuscle F-actin. Binding of PKC-
to actin was concentration dependent and enhanced by the presence of PKC activators. We also reported (15) for the first time that binding of PKC-
to actin is a necessary step toward activation of NKCC1. Actin binds to PKC-
at an NH2-terminal
C2-like domain. This domain encodes a 123-amino acid segment of the NH2-terminal region of PKC-
and, structurally, has antiparallel
-sheets with a P-type topology similar to phospholipase-
C2 domain (22). Our research revealed that the C2-like domain of PKC-
presents a novel phospholipid-independent binding site for interaction with nonmuscle actin. A recombinant
C2-like domain peptide blocked binding of PKC-
to nonmuscle actin and activation of NKCC1 by the
1-adrenergic agonist methoxamine. Thus the
C2-like domain likely expresses a specific binding site for nonmuscle F-actin that accounts for high-affinity binding of the two proteins, leading to activation of NKCC1.
The C2-like domain is analogous to the C2, or Ca2+-binding, domain of conventional PKC isotypes and may also share functional properties with the NH2 terminus of PKC-
(15). An eight-amino acid segment embedded in the V1, or NH2-terminal, region of PKC-
binds to receptor for activated C kinase 1 (RACK1) in Calu-3 cells and in cardiac myocytes (2, 16, 28). The purpose of our current study was to identify a site of F-actin binding on the
C2-like domain of PKC-
. Preliminary bioinformatic analysis indicated a unique
C2-like domain amino acid sequence that lacked homology to other actin-binding PKC isotypes or actin-associated proteins. Therefore, we prepared truncations of recombinant His6-tagged
C2-like domain for in vitro binding assays, in vivo functional evaluation of NKCC1 activation by methoxamine, and pulldown of actin from Calu-3 airway epithelial cells. The results of the study demonstrate inhibition of binding of PKC-
to nonmuscle F-actin by a proximal 17-amino acid segment at the NH2 terminus of the
C2-like domain. The truncated peptide blocked activation of NKCC1 by methoxamine and pulled down actin from total cell lysates of Calu-3 cells.
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METHODS |
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Expression and isolation of recombinant peptides.
Recombinant full-length C2-like domain and truncated peptides were produced with a pET-14b expression vector (Novagen), which placed a polyhistidine tag at the NH2 terminus of the construct. His6-tagged peptides were expressed in TOP10 One Shot-competent cells on induction with 0.1 mM isopropyl-
-D-1-thiogalactopyranoside for 4 h at 37°C. Cells were harvested, and recombinant protein was extracted from the bacterial cell pellet with a B-PER 6xHis spin purification kit (Pierce Biotechnology, Rockford, IL) and eluted in 2 ml of elution buffer. Peptides were purified with Centricon YM-3 extraction columns (Millipore), dialyzed against PBS, pH 7.4, and stored at 80°C. Protein content was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology) with bovine serum albumin as the standard. To verify the size of the peptide, the truncations were subjected to SDS gel electrophoresis on an 18% Tris·HCl gel under reducing conditions. Gels were stained for protein bands with GelCode Blue stain reagent (Pierce Biotechnology) and analyzed with a 6xHis protein tag stain (Pierce Biotechnology) or an INDIA His6 probe (Pierce Biotechnology) for the His6 tag.
Binding assay.
Each truncation was individually analyzed in solid-phase binding studies with nonmuscle -actin (Cytoskeleton, Denver, CO). Monomeric actin (G-actin) was stored in G-actin buffer consisting of (in mM) 5 Tris·HCl, pH 8.0, 0.2 ATP, 0.2 CaCl2, and 1.0
-mercaptoethanol at a final concentration of 100 µg/ml. On the day of use, G-actin was diluted to a concentration of 0.4 µg/µl in G-actin buffer. Filamentous actin (F-actin) was polymerized from G-actin by the addition of 1/10th volume of (in mM) 500 KCl, 20 MgCl2, and 10 ATP. The mixture was incubated for 1 h at room temperature and then diluted to 0.005 µg/µl in (in mM) 50 KCl, 2 MgCl2, and 1 ATP. Polymerized F-actin was vacuumed onto polyvinylidene difluoride (PVDF) membrane paper (Schleicher & Schuell) with a slot-blot apparatus. The membrane was blocked in F-actin buffer for 1 h, incubated with preactivated PKC-
with or without varying amounts of truncated peptide for 25 min, and washed extensively. PKC-
binding was detected by immunoblot analysis using a polyclonal antibody to PKC-
. As a positive control, membranes were reprobed for actin with a monoclonal antibody to
-actin.
Measurement of NKCC1 activity.
NKCC1 activity was measured as bumetanide-sensitive uptake of 86Rb, a congener of K, as previously described (16). Calu-3 cells were grown in submerged cell culture on 0.4-µm-pore Transwell-Clear polyester filter inserts and serum deprived for 24 h before experiments. His6-tagged full-length C2-like domain or truncated peptides were delivered into Calu-3 cells with a BioPORTER protein delivery system followed by measurement of bumetanide-sensitive 86Rb uptake. To initiate radiotracer uptake, filters were transferred to a well of a six-well tissue culture dish containing 1 µCi of 86Rb in HEPES-buffered HBSS (HPSS). Influx was measured for a 4-min time interval and then terminated by rapidly immersing filters four times in an ice-cold isotonic buffer consisting of 100 mM MgSO4 and 137 mM sucrose. Intracellular radioactivity was extracted by incubating cell monolayers in 0.1 N NaOH. Aliquots of extract were assayed for radioactive counts by liquid scintillation counting and for protein with a BCA protein assay kit (Pierce Biotechnology) using bovine serum albumin as the standard. Intracellular radioisotopic content was calculated as nanomoles of K per milligram of protein (86Rb).
Pulldown of endogenous actin.
Pulldowns were performed with His6-tagged full-length C2-like domain or truncated peptides. Total cell lysates from Calu-3 cells were prepared and incubated at room temperature for 30 min with 20 µg of recombinant protein. Talon beads were added to pull down His6-tagged peptide. Beads were recovered by centrifugation, washed extensively with PBS, and resuspended in Laemmli buffer. Samples were heated for 5 min in a boiling water bath, cooled, and then subjected to 415% SDS-PAGE and immunoblot analysis for F-actin with a monoclonal antibody to actin. Endogenous actin was detected as a 45-kDa protein band.
Data analysis. Immunoreactive protein bands were quantitated with a VersaDoc Imaging System (Bio-Rad, Hercules, CA) or laser densitometry. Data are representative of three or more experiments and are reported as means ± SE. Treatment effects were evaluated with a two-sided Student's t-test. An inhibitory constant or IC50 was calculated from Hill plots of data from dose-response curves with a GraphPad Prism 4.0 software program.
Materials.
86Rb (specific activity 154 Bq/g Rb, 4,200 Ci/g Rb) was purchased from Amersham Life Science. Restriction enzymes, Klenow polymerase, and T4 DNA ligase were obtained from New England Biolabs. Polyclonal anti-PKC- antibody and horseradish peroxidase-coupled secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Baculovirus-expressed recombinant PKC-
was obtained from Pan Vera (Madison, WI) and monoclonal antibody to
-actin and nonmuscle actin (85%
-actin, 15%
-actin) were from Cytoskeleton. The BioPORTER protein delivery system was obtained from Gene Therapy Systems (San Diego, CA). Methoxamine HCl and bumetanide were purchased from Sigma (St. Louis, MO), 18% precast slab gels from Bio-Rad, and protease inhibitor cocktail set III from Calbiochem-Novabiochem (San Diego, CA). An enhanced chemiluminescence reagent was purchased from Amersham (Piscataway, NJ) and Transwell Clear filter inserts from Fisher Scientific (Hanover Park, IL). INDIA His6 probe and Supersignal West Pico Chemiluminescent substrate were purchased from Pierce Biotechnology. Agarose beads and tissue culture supplies were purchased from Invitrogen-GIBCO (Gaithersburg, MD). All other chemicals were reagent grade.
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RESULTS |
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Pulldown of endogenous actin by His6-tagged peptides.
If the C2-like domain encodes a binding site for F-actin, we predict that the peptide will bind to endogenous F-actin. To examine this possibility, we used His6-tagged full-length
C2-like domain or truncated peptides in a pulldown assay using total cell lysate derived from Calu-3 cells. The results are shown in Fig. 5. Full-length
C2-like domain pulled down 27.8% of total actin detected in Calu-3 cell lysates. N2, N4, and N6 truncated peptides pulled down 21.5%, 25.2%, and 24.6% of total actin, respectively. C1, C3, and C5 truncated peptides pulled down 5.7%, 6.2% and 6.2% of total actin, respectively. The fourfold greater recovery of actin with the NH2-terminal peptides indicates preferential association of endogenous F-actin with the NH2 terminus of PKC-
.
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DISCUSSION |
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F-actin is a recognized binding partner of PKC isotypes and, as such, is thought to selectively position a PKC isotype in an appropriate intracellular location to respond to specific receptor-mediated activation signals. Disruption of methoxamine-stimulated NKCC1 activation by the C2-like domain (15) and by NH2-terminal truncated peptides (Fig. 4) indicates that binding of PKC-
to F-actin is necessary to maximize the hormonal response. Two other PKC isotypes, PKC-
II and PKC-
, bind directly to F-actin via specific domains (Table 1). PKC-
II interacts with F-actin in in vitro assays and coimmunoprecipitates actin through a specific actin binding site on the COOH terminus (1). The COOH terminus of PKC-
II shares homology with a consensus actin-binding site in troponin (Table 1) and effectively blocks binding of PKC-
II and F-actin. PKC-
directly localizes with and binds F-actin at a binding site mapped to amino acids 223228 in the C1 domain of the NH2 terminus regulatory domain of PKC-
(26). This binding motif has also been identified in several other actin-binding proteins, such as
-actinin, actobindin, fimbrin, myosin, plastin, and thymosin (Table 1). A hexapeptide (LKKQET) based on the PKC-
actin-binding motif competes in a dose-dependent manner with binding of PKC-
to F-actin (26). A computer analysis of the actin-binding domains shows that the actin-binding domain on the
C2-like region of PKC-
is distinct from actin binding sites on PKC-
II and PKC-
and also from actin binding sites of other recognized actin-binding proteins, such as annexin and ezrin (Table 1).
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Structurally, the NH2-terminal sequence defining the C2-like domain resembles the C2 domains of PLC
and synaptotagmin I (9, 22). The crystal structure of
C2-like domain reveals an antiparallel
-sandwich structure with a P-type topology similar to PLC
C2 domain. Two
-sheets are formed by the
1-,
4-,
7-, and
8-strands and the
2-,
5-, and
6-strands. The first 17 amino acids of the
C2-like domain comprise a
1-strand plus 4 amino acids of the 13-residue
1
2 loop. Our data indicate direct binding of the
1-strand to nonmuscle F-actin with functional consequences on the activation of NKCC1. The remainder of the
C2-like domain is thought to express COOH-terminal loops equivalent to the Ca2+-binding region of a C2 domain and two potential lipid binding sites (22). The C2 domain of conventional PKC isotypes facilitates Ca2+-dependent phospholipid binding with the lipid phosphatidylserine through electrostatic binding of Ca2+ (8, 13, 24, 34, 33). The
C2-like domain, on the other hand, has analogous Ca2+-binding loops that lack all but one of five conserved Ca2+-coordinating side chains and hence are unable to bind Ca2+.
The function of the C2-like domain, although not fully understood, differs markedly from that of the C2 domain in Ca2+-dependent conventional PKC isotypes. C2-like domains are thought to be important for translocation of PKC isotypes from cytosol to membrane-bound anchor or scaffold proteins, thus playing a critical role in localizing the activated PKC enzyme near its target substrate. In neutrophils, PKC- colocalizes with F-actin in a PMA-sensitive manner; colocalization is blocked by the PKC-
inhibitor rottlerin (19). Exposure of neutrophils to Staphylococcus aureus causes a rapid and dramatic redistribution of PKC-
and F-actin. In the same study, microinjected
C2-like domains colocalized with F-actin and prevented its redistribution. These findings indicate an interaction of PKC-
with an active pool of actin that is necessary for F-actin redistribution. However, the observation that not all cellular PKC-
interacts with cellular actin and not all F-actin colocalizes with PKC-
mirrors our findings (15) from immunofluorescence and confocal microscopy of PKC-
interaction with F-actin in Calu-3 airway epithelial cells. Indeed, the interaction of PKC-
and F-actin appears to sequester PKC-
to specific intracellular regions and, in Calu-3 cells, is necessary for activation of NKCC1.
How the interaction between PKC- and nonmuscle actin regulates NKCC1 function remains to be clarified. Anchoring of activated PKC-
to F-actin might align PKC-
with a target substrate for phosphorylation or sequester PKC-
in a proteome that regulates NKCC1 activity. Understanding the relationship among PKC-
, the actin cytoskeleton, and NKCC1 will clearly enhance our understanding of NKCC1 function and hence will be the focus of future research.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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