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Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca2+ Stores

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Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca2+ Stores UCSF UC San Francisco Previously Published Works Title Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores Permalink https://escholarship.org/uc/item/5374h7q6 Journal Journal of Investigative Dermatology, 121(4) ISSN 0022-202X Authors Behne, M J Tu, Chia-Ling L Aronchik, I et al. Publication Date 2003-10-01 Peer reviewed eScholarship.org Powered by the California Digital Library University of California ORIGINAL ARTICLE Human Keratinocyte ATP2C1 Localizes to the Golgi and Controls Golgi Ca2 þ Stores Martin J. Behne,Ãw Chia-Ling Tu,z Ida Aronchik,w Ervin Epstein,Ãy Graham Bench,8 Daniel D. Bikle,z Tullio Pozzan,z and Theodora M. MauroÃw ÃDepartment of Dermatology, University of California, San Francisco, California, USA; wDermatology Service and zDepartment of Medicine, Endocrine Unit,VA Medical Center, San Francisco, California, USA; yDepartment of Dermatology, San Francisco General Hospital, San Francisco, California, USA; 8Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California, USA; zDepartment of Biomedical Sciences and Consiglio Nazionale delle Ricerche (CNR) Center for the Study of Biomembranes, University of Padova, 35121 Padova, Italy Hailey^Hailey disease (MIM16960) is a blistering skin parable to other epithelial cells, Hailey^Hailey disease disease caused by mutations in the Ca2 þ ATPase keratinocyte Golgi Ca2 þ re¢ll is slower, and the maxi- ATP2C1. We found that the abnormal Ca2 þ signaling mum Ca2 þ concentration reached is signi¢cantly lower. seen in Hailey^Hailey disease keratinocytes correlates with These ¢ndings were replicated in vivo, because clinically decreased protein levels of ATP2C1. Human ATP2C1 normal Hailey^Hailey disease epidermis contained low- protein approximated 115 kDa in size. The ATP2C1 er Ca2 þ stores and displayed an abnormal Ca2 þ gradi- is localized to the Golgi apparatus in human keratino- ent. In this report we localize the ATP2C1, demonstrate cytes, similar to its localization in yeast and Caenorhab- its physiologic relevance in mammalian cells, and ditis elegans. To test whether the ATP2C1 controls Golgi measure intraorganelle Golgi Ca2 þ in keratinocytes. Ca2 þ stores, we measured intraorganelle Ca2 þ concen- Key words: Hailey^Hailey disease/aequorin/adhesion/epider- trations using speci¢cally targeted aequorins. Whereas mal permeability barrier. J Invest Dermatol 121:688 ^694, 2003 normal keratinocytes display Golgi Ca2 þ levels com- ailey-Hailey disease (HHD) is an autosomal domi- (Hu et al, 2000), suggesting that ATP2C1 controls a Ca2 þ store nant blistering skin disease, characterized histo- that is important in the keratinocyte response to raised extracellu- logically by defects in cell-to-cell adhesion in lar Ca2 þ. the suprabasal layers of the epidermis (acantholysis). Although the central role of the PMR1 and Golgi calcium HHD is a result of mutations in ATP2C1 (Hu et al, stores is well established in yeast (Sorin et al, 1997; Durr et al, H2000; Sudbrak et al,2000),aCa2 þ ATPase localized to the Golgi 1998), the discovery that ATP2C1 mutations caused defects in in Caenorhabditis elegans (Van Baelen et al, 2001) and whose homo- both intracellular Ca2 þ sequestration and intracellular Ca2 þ sig- log, PMR1, also is localized to the Golgi in yeast (Antebi and naling, and resulted in human disease, was the ¢rst indication of Fink, 1992). Histologic and immuno£uorescence studies of the importance of this Ca2 þ ATPase in mammals (Hu et al,2000; HHD demonstrate internalization of desmosomal components, Sudbrak et al, 2000). Mutations found in HHD patients most of- whereas adherens junctions and gap junctions are preserved (Har- ten predict prematurely truncated products through frameshift ada et al, 1994; Hashimoto et al, 1995; Metze et al, 1996). In normal mutations or single-base-pair substitutions, consistent with a keratinocytes in vitro,Ca2 þ stimulates the assembly of functional haploinsu⁄ciency pathogenesis (Hu et al, 2000). Endoplasmic re- desmosomes, both by causing the formation of actin ¢lopodia ticulum (ER) calcium stores also are present in keratinocytes, and and by complexing desmosomal components including E-cad- pharmacologic blockade of the Ca2 þ ATPase ATP2A2 with herin, a-, b-, and g-catenin (Vasioukhin et al,2000).Raisedex- thapsigargin (TG) prevents Ca2 þ -induced redistribution of E- tracellular Ca2 þ acts through a well-de¢ned cascade of signaling cadherin to the cell periphery (Li et al, 1995). Mutations in the events, including binding to a plasma membrane Ca2 þ receptor, ATP2A2 cause Darier’s disease (Sakuntabhai et al, 1999), a similar generation of inositol 1,4,5-trisphosphate (IP3), release of intracel- blistering skin disease. In contrast, the role of ATP2C1-controlled lular Ca2 þ, and subsequent in£ux of Ca2 þ through plasma Golgi Ca2 þ stores in keratinocyte Ca2 þ -sensitive processes such membrane channels. HHD keratinocytes do not release intra- as growth, di¡erentiation, and adhesion has not yet been de¢ned. cellular Ca2 þ when stimulated with raised extracellular Ca2 þ The yeast homolog to the ATP2C1, PMR1, localizes to the med- ial Golgi (Sorin et al, 1997) and can raise Golgi Ca2 þ concentra- tions to levels as high as 1 mM (Taylor et al, 1997). In yeast, Manuscript received November 5, 2002; revised February 17, 2003; intraorganelle Ca2 þ controls Golgi transport (Carnell and accepted for publication May 12, 2003 Moore, 1994), N-linked glycosylation, accurate sorting of car- Reprint requests to: Martin J. Behne, Dermatology Service (190), Veter- Ã ans A¡airs Medical Center, 4150 Clement Street, San Francisco, CA 94121. boxypeptidase Y, and the appropriate degradation of Cyp , Email: [email protected] a misfolded soluble ER protein (Durr et al, 1998). PMR1 mutants are intolerant to raised extracellular Ca2 þ and demonstrate Abbreviations: ER, endoplasmic reticulum; HHD, Hailey^Hailey dis- 2 þ ease; IP3, inositol 1,4,5-trisphosphate KRB, Krebs^Ringer bicarbonate; higher cytosolic Ca concentrations (Halachmi and Eilam, TG, thapsigargin; PIXE, proton-induced X-ray emission (analysis). 1996), similar to that seen in HHD keratinocytes (Hu et al,2000). 0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 688 VOL. 121, NO. 4 OCTOBER 2003 ATP2C1 CONTROLS GOLGI CA2 þ 689 Experiments demonstrating TG-sensitive and -insensitive cellular Ca2 þ. Calibration was performed at the end of each experiment by lysing þ stores suggested that a Ca2 ATPase distinct from the ATP2A2 the cells with a solution containing 100 mM digitonin and 10 mM CaCl2 in 2 þ H O. The photomultiplier output was ampli¢ed by a built-in ampli¢er^ was linked to IP3 -sensitive Ca pools (Pizzo et al, 1997). These 2 ¢ndings were con¢rmed and expanded by the discovery that the discriminator. This signal was captured by a Thorn-EMI photon counting board and stored in an IBM-compatible computer for further analysis. The homologous PMR1 ortholog expressed in C. elegans controls an 2 þ 2 þ resulting luminescence data were calibrated into Ca values, using a IP3 -sensitive Ca store located in the Golgi (Van Baelen et al, computer algorithm (Brini et al, 1995). 2001). Human ATP2C1 is homologous but not identical to yeast PMR1 (Rudolph et al, 1989) and encodes two alternatively spliced transcripts, ATP2C1a and ATP2C1b. Cellular fractionation (sucrose gradient) Normal keratinocytes 2 þ were cultured as above. The cells were homogenized using a Polytron Unlike measurements of cytosolic Ca , Golgi intraorganelle homogenizer, the nuclei removed by 500 Â g centrifugation, and crude 2 þ 2 þ Ca cannot be measured accurately with Ca -sensitive dyes. membranes were collected by high-speed centrifugation at 10,000 Â g. The Therefore, we used a targeted aequorin approach similar to that resulting supernatant was layered on a discontinuous, multilayer sucrose used to study the Ca2 þ concentrations in mitochondria (Rizzuto gradient (15%^50%), centrifuged in a Beckman SW28 rotor at 100,000 et al, 1992), nucleus (Brini et al, 1993), and ER (Montero et al,1995). Â g for 16 h, and the membranous materials in the various sucrose layers We transfected normal and HHD keratinocytes with a chimeric collected for western blot analysis. Organelle and ATP2C1 localization was cDNA that encodes an HA1-tagged aequorin (Brini et al,1995) accomplished using antibodies to BiP (mouse monoclonal antibody and the transmembrane portion of sialyltransferase, which targets 610978, BD Transduction Laboratories, San Diego, CA) as the ER marker, the aequorin chimera to the Golgi (Pinton et al,1998).Thistool, GM130 as the cis-Golgi marker (mouse monoclonal antibody 610822, BD - Transduction Laboratories), p230 (mouse monoclonal antibody 611280, BD and its related aequorin mutant (Asp119 Ala), which converts Transduction Laboratories), and TGN38 (mouse monoclonal antibody the aequorin to one with a lower Ca2 þ a⁄nity and thus a more 2 þ 610898, BD Transduction Laboratories) as the trans-Golgi markers and long-lasting signal at micromolar Ca concentrations, has been ATP2C1 (PMR1, rabbit polyclonal IgG, sc-5548, Santa Cruz Bio- used previously to measure Golgi Ca2 þ concentrations in normal technology, Santa Cruz, CA) as well as ATP2A2 (SERCA2, sc-8094, and Bcl-2-transfected HeLa cells (Pinton et al, 1998; Pinton et al, Santa Cruz Biotechnology). 2000). Using this approach, we found that normal human kerati- 2 þ nocytes also sequester substantial Ca pools into the Golgi ap- Immunohistochemistry Normal keratinocytes, cultured as described paratus and that these stores are depleted in keratinocytes and above, were ¢xed with 4% formaldehyde in phosphate-bu¡ered saline for epidermis after mutation of the ATP2C1. Further, we have loca- 10 min. ATP2C1 was labeled using a rabbit polyclonal antibody to human lized the ATP2C1 protein to the Golgi apparatus in keratinocytes, ATP2C1 (sc-5548, Santa Cruz Biotechnology). The same cells also were using cellular fractionation and functional Ca2 þ concentration labeled with the trans-Golgi membrane-associated protein p230 (mouse studies.
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