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Desmoyokin Still Goes On
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A Mystery of AHNAK/Desmoyokin Still Goes On Masayuki Amagai Department of Dermatology, Keio University School of Medicine, Tokyo, Japan
Ca þ 2 channel through the C-terminal region (aa 5262– 5643), suggesting its role in the subsarcolemmal cytoarchitecture as a linker protein between cardiac L-type Ca2 þ channels and the actin-based cytoskeleton (Hohaus et al, 2002). At the plasma membrane, AHNAK/desmoyokin interacts with the annexin 2/S100A10 complex and regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture (Benaud et al, 2004). All of these experimental results are based on the findings in vitro, using cultured cells. The biological function of AHNAK/desmoyokin in vivo remains to be elucidated. In this issue of the JID, Kouno et al (2004) generated AHNAK/desmoyokin-deficient mice by gene-targeting technique. The AHNAK/desmoyokin-deficient ES cells proliferated with similar doubling time to wild-type cells and showed the normal developmental potential to differentiate into several lineages including cardiac cells and neural cells. AHNAK/desmoyokin-deficient mice were viable and fertile and showed no gross developmental defects or macroscopic abnormalities. Histologically, skin sections of AHNAK/desmoyokin-deficient mice at different ages during appendage development showed no abnormalities of hair follicles and sebaceous glands. By immunohistochemistry, mutant epidermal cells expressed the epidermal differentiation markers laminin 5, keratin 5, keratin 1, and involucrin, as found in normal epidermis. The mutant epidermis treated with 12o-tetradecanoyphorbol-13-acetate (TPA), which stimulates protein kinase C and induces the translocation of AHNAK/ desmoyokin, showed similar increased numbers of BrdUincorporated cells and similar thickness of the epidermis as found in TPA-treated normal epidermis. Electron microscopic studies revealed no abnormality in the ultrastructure of intercellular junctions or in the localization of desmocollin, a transmembrane component of desmosomes. There was no spontaneous tumor formation in the mutant mice, and twostage carcinogenesis experiments showed no significant difference in tumor formation rates between wild-type and the mutant mice. Taken together, the authors concluded that the deficiency of AHNAK/desmoyokin in mice has only minimal, if any, effects on epidermal cell adhesion, tumorigenesis, cell proliferation, and differentiation. Why no phenotype? No phenotype of mice deficient for a certain gene indicates that other molecules compensate for the loss of function caused by the defected gene product. Just recently, Benaud and his colleagues used small interfering RNA or siRNA, another method to see the effect by the loss of function, and demonstrated that downregulation of AHNAK/desmoyokin using specific siRNA prevented cortical actin cytoskeleton reorganization that is required to support cell height in Madin-Darby canine kidney cells
The recombinant technique for the development of gene knockout mice has played a critical role in clarifying the biological in vivo functions of the proteins of interest. This technique is powerful, but it requires labor-intensive and time-consuming processes. Therefore, gene target projects have a certain risk, especially for the young fellows who often actually do the work. It requires multiple steps: characterization of genomic sequences, construction of targeted vector, screening of embryonic stem (ES) cells with homologous recombination, injection of ES clones to blastocysts, screening for mutant mice, etc. The real difficulty, however, may start when the knockout mice do not have any apparent phenotype. Desmoyokin was originally identified as a new desmosomal plaque protein that is found at the periphery of the cytoplasmic plaque of desmosomes in the stratified squamous epithelia (Hieda and Tsukita, 1989). Hashimoto and his colleagues isolated mouse monoclonal antibodies against a desmosome preparation made from bovine muzzle epidermis and found that one of them recognized desmoyokin. Using the monoclonal antibody as an antibody probe, they immunoscreened a mouse keratinocyte expression cDNA library and isolated cDNA clones encoding desmoyokin (Hashimoto et al, 1993). The deduced amino acid sequence revealed that desmoyokin is identical to AHNAK, which is a tumor-related protein of exceptionally large size (700 kDa) and downregulated in cell lines of neuroblastoma, small cell lung carcinomas, and Burkitt lymphomas (Shtivelman et al, 1992). AHNAK is encoded by an intronless gene located on human chromosome 11q12–13 and has three main structural regions: the N-terminal 251 amino acids, a large central region of 4390 amino acids with multiple repeated units of about 128 amino acids in length, and the C-terminal of 1002 amino acids. The central region is predicted to have antiparallel b-strands connected by intervening loops. Several putative regulatory elements are clustered within the C-terminal region, including nuclear export localization signals, a leucine zipper, and potential phosphorylation sites for protein kinase B and C (Nie et al, 2000; Sussman et al, 2001). AHNAK/desmoyokin is a ubiquitous protein expressed in a variety of cell types. In epithelial cells AHNAK/desmoyokin is distributed mainly on the cell membranes, suggesting its role in cell–cell adhesion. On the other hand, AHNAK/ desmoyokin is expressed in the nucleus and cytoplasm of nonepithelial cells. It also shuttles between nucleus and cytoplasm depending on extracellular calcium concentration, and the C-terminus is responsible for this translocation (Nie et al, 2000). In cardiomyocytes, AHNAK/desmoyokin is associated with the regulatory b2 subunit of the cardiac L-type
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
xiv
123 : 4 OCTOBER 2004
(Benaud et al, 2004). siRNA specific for AHNAK/desmoyokin could interfere with not only AHNAK/desmoyokin but also other proteins containing similar sequences, such as titin, which has a PEVK region similar to the central repeated sequence of AHNAK/desmoyokin (Wernyj et al, 2001). Furthermore, a search for genes with homologous sequences with AHNAK/desmoyokin in the human genome sequence database found the second AHNAK-like protein, AHNAK2, on chromosome 14q32 (Komuro et al, 2004). AHNAK2 is a 600 kD protein composed of a large number of highly conserved repeat segments, that may have a series of linked, antiparallel b-strands like AHNAK/desmoyokin. AHNAK/ desmoyokin-deficient mice maintained the staining with polyclonal antibodies directed against epitopes specific for AHNAK/desmoyokin, suggesting that AHNAK/desmoyokin and AHNAK2 may be coexpressed in the same cell (Komuro et al, 2004). Therefore, it is also speculated that ANHAK2 compensates the loss of function of AHNAK/desmoyokin and, therefore, AHNAK/desmoyokin-deficient mice show no obvious phenotype. Thus, the biological in vivo function of AHNAK/desmoyokin remains to be elucidated and the journey toward a better understanding of this mysterious molecule still goes on.
DOI: 10.1111/j.0022-202X.2004.23432.x
MYSTERY OF AHNAK/DESMOYOKIN
xv
References Benaud C, Gentil BJ, Assard N, Court M, Garin J, Delphin C, Baudier J: AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture. J Cell Biol 164:133–144, 2004 Hashimoto T, Amagai M, Parry DAD, et al: Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK. J Cell Sci 105:275–286, 1993 Hieda Y, Tsukita S: A new high molecular mass protein showing unique localization in desmosomal plaque. J Cell Biol 109:1511–1518, 1989 Hohaus A, Person V, Behlke J, Schaper J, Morano I, Haase H: The carboxylterminal region of ahnak provides a link between cardiac L-type Ca þ 2 channels and the actin-based cytoskeleton. FASEB J 16:1205–1216, 2002 Komuro A, Masuda Y, Kobayashi K, Babbitt R, Gunel M, Flavell RA, Marchesi VT: The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells. Proc Natl Acad Sci USA 101:4053–4058, 2004 Kouno M, Kondoh G, Horie K, et al: AHNAK/desmoyokin is dispensable for proliferation, differentiation, and maintenance of integrity in mouse epidermis. J Invest Dermatol 123:700–707, 2004 Nie Z, Ning W, Amagai M, Green KJ, Hashimoto T: C-terminus of desmoyokin/ AHNAK protein is responsible for its translocation between the nucleus and cytoplasm. J Invest Dermatol 114:1044–1049, 2000 Shtivelman E, Cohen FE, Bishop JM: A human gene (AHNAK) encoding an unusually large protein with a 1.2-microns polyionic rod structure. Proc Natl Acad Sci USA 89:5472–5476, 1992 Sussman J, Stokoe D, Ossina N, Shtivelman E: Protein kinase B phosphorylates AHNAK and regulates its subcellular localization. J Cell Biol 154: 1019–1030, 2001 Wernyj RP, Ewing CM, Isaacs WB: Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery. Cell Motil Cytoskeleton 50:101–113, 2001
A Mystery of AHNAK/Desmoyokin Still Goes On Masayuki Amagai Department of Dermatology, Keio University School of Medicine, Tokyo, Japan
Ca þ 2 channel through the C-terminal region (aa 5262– 5643), suggesting its role in the subsarcolemmal cytoarchitecture as a linker protein between cardiac L-type Ca2 þ channels and the actin-based cytoskeleton (Hohaus et al, 2002). At the plasma membrane, AHNAK/desmoyokin interacts with the annexin 2/S100A10 complex and regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture (Benaud et al, 2004). All of these experimental results are based on the findings in vitro, using cultured cells. The biological function of AHNAK/desmoyokin in vivo remains to be elucidated. In this issue of the JID, Kouno et al (2004) generated AHNAK/desmoyokin-deficient mice by gene-targeting technique. The AHNAK/desmoyokin-deficient ES cells proliferated with similar doubling time to wild-type cells and showed the normal developmental potential to differentiate into several lineages including cardiac cells and neural cells. AHNAK/desmoyokin-deficient mice were viable and fertile and showed no gross developmental defects or macroscopic abnormalities. Histologically, skin sections of AHNAK/desmoyokin-deficient mice at different ages during appendage development showed no abnormalities of hair follicles and sebaceous glands. By immunohistochemistry, mutant epidermal cells expressed the epidermal differentiation markers laminin 5, keratin 5, keratin 1, and involucrin, as found in normal epidermis. The mutant epidermis treated with 12o-tetradecanoyphorbol-13-acetate (TPA), which stimulates protein kinase C and induces the translocation of AHNAK/ desmoyokin, showed similar increased numbers of BrdUincorporated cells and similar thickness of the epidermis as found in TPA-treated normal epidermis. Electron microscopic studies revealed no abnormality in the ultrastructure of intercellular junctions or in the localization of desmocollin, a transmembrane component of desmosomes. There was no spontaneous tumor formation in the mutant mice, and twostage carcinogenesis experiments showed no significant difference in tumor formation rates between wild-type and the mutant mice. Taken together, the authors concluded that the deficiency of AHNAK/desmoyokin in mice has only minimal, if any, effects on epidermal cell adhesion, tumorigenesis, cell proliferation, and differentiation. Why no phenotype? No phenotype of mice deficient for a certain gene indicates that other molecules compensate for the loss of function caused by the defected gene product. Just recently, Benaud and his colleagues used small interfering RNA or siRNA, another method to see the effect by the loss of function, and demonstrated that downregulation of AHNAK/desmoyokin using specific siRNA prevented cortical actin cytoskeleton reorganization that is required to support cell height in Madin-Darby canine kidney cells
The recombinant technique for the development of gene knockout mice has played a critical role in clarifying the biological in vivo functions of the proteins of interest. This technique is powerful, but it requires labor-intensive and time-consuming processes. Therefore, gene target projects have a certain risk, especially for the young fellows who often actually do the work. It requires multiple steps: characterization of genomic sequences, construction of targeted vector, screening of embryonic stem (ES) cells with homologous recombination, injection of ES clones to blastocysts, screening for mutant mice, etc. The real difficulty, however, may start when the knockout mice do not have any apparent phenotype. Desmoyokin was originally identified as a new desmosomal plaque protein that is found at the periphery of the cytoplasmic plaque of desmosomes in the stratified squamous epithelia (Hieda and Tsukita, 1989). Hashimoto and his colleagues isolated mouse monoclonal antibodies against a desmosome preparation made from bovine muzzle epidermis and found that one of them recognized desmoyokin. Using the monoclonal antibody as an antibody probe, they immunoscreened a mouse keratinocyte expression cDNA library and isolated cDNA clones encoding desmoyokin (Hashimoto et al, 1993). The deduced amino acid sequence revealed that desmoyokin is identical to AHNAK, which is a tumor-related protein of exceptionally large size (700 kDa) and downregulated in cell lines of neuroblastoma, small cell lung carcinomas, and Burkitt lymphomas (Shtivelman et al, 1992). AHNAK is encoded by an intronless gene located on human chromosome 11q12–13 and has three main structural regions: the N-terminal 251 amino acids, a large central region of 4390 amino acids with multiple repeated units of about 128 amino acids in length, and the C-terminal of 1002 amino acids. The central region is predicted to have antiparallel b-strands connected by intervening loops. Several putative regulatory elements are clustered within the C-terminal region, including nuclear export localization signals, a leucine zipper, and potential phosphorylation sites for protein kinase B and C (Nie et al, 2000; Sussman et al, 2001). AHNAK/desmoyokin is a ubiquitous protein expressed in a variety of cell types. In epithelial cells AHNAK/desmoyokin is distributed mainly on the cell membranes, suggesting its role in cell–cell adhesion. On the other hand, AHNAK/ desmoyokin is expressed in the nucleus and cytoplasm of nonepithelial cells. It also shuttles between nucleus and cytoplasm depending on extracellular calcium concentration, and the C-terminus is responsible for this translocation (Nie et al, 2000). In cardiomyocytes, AHNAK/desmoyokin is associated with the regulatory b2 subunit of the cardiac L-type
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
xiv
123 : 4 OCTOBER 2004
(Benaud et al, 2004). siRNA specific for AHNAK/desmoyokin could interfere with not only AHNAK/desmoyokin but also other proteins containing similar sequences, such as titin, which has a PEVK region similar to the central repeated sequence of AHNAK/desmoyokin (Wernyj et al, 2001). Furthermore, a search for genes with homologous sequences with AHNAK/desmoyokin in the human genome sequence database found the second AHNAK-like protein, AHNAK2, on chromosome 14q32 (Komuro et al, 2004). AHNAK2 is a 600 kD protein composed of a large number of highly conserved repeat segments, that may have a series of linked, antiparallel b-strands like AHNAK/desmoyokin. AHNAK/ desmoyokin-deficient mice maintained the staining with polyclonal antibodies directed against epitopes specific for AHNAK/desmoyokin, suggesting that AHNAK/desmoyokin and AHNAK2 may be coexpressed in the same cell (Komuro et al, 2004). Therefore, it is also speculated that ANHAK2 compensates the loss of function of AHNAK/desmoyokin and, therefore, AHNAK/desmoyokin-deficient mice show no obvious phenotype. Thus, the biological in vivo function of AHNAK/desmoyokin remains to be elucidated and the journey toward a better understanding of this mysterious molecule still goes on.
DOI: 10.1111/j.0022-202X.2004.23432.x
MYSTERY OF AHNAK/DESMOYOKIN
xv
References Benaud C, Gentil BJ, Assard N, Court M, Garin J, Delphin C, Baudier J: AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture. J Cell Biol 164:133–144, 2004 Hashimoto T, Amagai M, Parry DAD, et al: Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK. J Cell Sci 105:275–286, 1993 Hieda Y, Tsukita S: A new high molecular mass protein showing unique localization in desmosomal plaque. J Cell Biol 109:1511–1518, 1989 Hohaus A, Person V, Behlke J, Schaper J, Morano I, Haase H: The carboxylterminal region of ahnak provides a link between cardiac L-type Ca þ 2 channels and the actin-based cytoskeleton. FASEB J 16:1205–1216, 2002 Komuro A, Masuda Y, Kobayashi K, Babbitt R, Gunel M, Flavell RA, Marchesi VT: The AHNAKs are a class of giant propeller-like proteins that associate with calcium channel proteins of cardiomyocytes and other cells. Proc Natl Acad Sci USA 101:4053–4058, 2004 Kouno M, Kondoh G, Horie K, et al: AHNAK/desmoyokin is dispensable for proliferation, differentiation, and maintenance of integrity in mouse epidermis. J Invest Dermatol 123:700–707, 2004 Nie Z, Ning W, Amagai M, Green KJ, Hashimoto T: C-terminus of desmoyokin/ AHNAK protein is responsible for its translocation between the nucleus and cytoplasm. J Invest Dermatol 114:1044–1049, 2000 Shtivelman E, Cohen FE, Bishop JM: A human gene (AHNAK) encoding an unusually large protein with a 1.2-microns polyionic rod structure. Proc Natl Acad Sci USA 89:5472–5476, 1992 Sussman J, Stokoe D, Ossina N, Shtivelman E: Protein kinase B phosphorylates AHNAK and regulates its subcellular localization. J Cell Biol 154: 1019–1030, 2001 Wernyj RP, Ewing CM, Isaacs WB: Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery. Cell Motil Cytoskeleton 50:101–113, 2001