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Kazrin, a novel periplakin-interacting protein associated with desmosomes and the keratinocyte plasma membrane.

Groot KR, Sevilla LM, Nishi K, DiColandrea T, Watt FM - J. Cell Biol. (2004)

Bottom Line: Kazrin is expressed in all layers of stratified squamous epithelia; it becomes membrane associated in the suprabasal layers, coincident with up-regulation of periplakin, and is incorporated into the cornified envelope of cultured keratinocytes.On transfection, all three kazrin isoforms have similar subcellular distributions.We conclude that kazrin is a novel component of desmosomes that associates with periplakin.

View Article: PubMed Central - PubMed

Affiliation: Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

ABSTRACT
Periplakin forms part of the scaffold onto which the epidermal cornified envelope is assembled. The NH2-terminal 133 amino acids mediate association with the plasma membrane and bind a novel protein, kazrin. Kazrin is highly conserved and lacks homology to any known protein. There are four alternatively spliced transcripts, encoding three proteins with different NH2 termini. Kazrin is expressed in all layers of stratified squamous epithelia; it becomes membrane associated in the suprabasal layers, coincident with up-regulation of periplakin, and is incorporated into the cornified envelope of cultured keratinocytes. Kazrin colocalizes with periplakin and desmoplakin at desmosomes and with periplakin at the interdesmosomal plasma membrane, but its subcellular distribution is independent of periplakin. On transfection, all three kazrin isoforms have similar subcellular distributions. We conclude that kazrin is a novel component of desmosomes that associates with periplakin.

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Kazrin and its interaction with periplakin. (A) Intron-exon organization of the kazrin gene, drawn to scale. Arrows, translation initiation sites; diagonal lines, 320 kb gap; π, leucine zipper-like domain; black bar spanning exons 3 to 5, α-helical region; asterisk, putative NLS sequence. TGA, translation termination codon; AATAAA, polyadenylation signal. Sequence data available from GenBank/EMBL/DDBJ under accession no. AY505119, AY505120, AY505121, and AY505122, corresponding to kazrin isoform a, b, c, and d, respectively. (B) Secondary structure predictions of kazrin C, based on algorithms of Chou/Fasman and Garnier/Robson, and two-hybrid analysis of interaction between P133 and kazrin deletion constructs. Numbers correspond to amino acids of kazrin C. Interactions scored positive if yeast grew on selective medium. (C) Immunoblots of GST pull-downs from keratinocyte lysates with immobilized GST-kazrin A (KA), GST-kazrin B (KB), GST-kazrin C (KC), GST-kazrin C aa 49–327 (KCΔ), or GST alone (GST). Blots were probed with antibodies specific for periplakin (Ppl; TD2), envoplakin (Evpl; CR5), or desmoplakin (Dp; 115F). As a control, 4% of cell lysate used in the pull-downs was loaded on gel (Extr). (D) Coimmunoprecipitation of kazrin C with P133. Cos-7 cells were transiently transfected with kazrin C-FLAG (K) together with empty vector (V) or P133-HA (P). Proteins were immunoprecipitated with anti-HA Y11 (HA IP), and immunoblots were probed with anti-FLAG mAb M2. As a control, 5% of cell lysate used in the immunoprecipitation was run in parallel (lysate). Positions of kazrin (K) and immunoglobulin light chain (Ig) are indicated.
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fig2: Kazrin and its interaction with periplakin. (A) Intron-exon organization of the kazrin gene, drawn to scale. Arrows, translation initiation sites; diagonal lines, 320 kb gap; π, leucine zipper-like domain; black bar spanning exons 3 to 5, α-helical region; asterisk, putative NLS sequence. TGA, translation termination codon; AATAAA, polyadenylation signal. Sequence data available from GenBank/EMBL/DDBJ under accession no. AY505119, AY505120, AY505121, and AY505122, corresponding to kazrin isoform a, b, c, and d, respectively. (B) Secondary structure predictions of kazrin C, based on algorithms of Chou/Fasman and Garnier/Robson, and two-hybrid analysis of interaction between P133 and kazrin deletion constructs. Numbers correspond to amino acids of kazrin C. Interactions scored positive if yeast grew on selective medium. (C) Immunoblots of GST pull-downs from keratinocyte lysates with immobilized GST-kazrin A (KA), GST-kazrin B (KB), GST-kazrin C (KC), GST-kazrin C aa 49–327 (KCΔ), or GST alone (GST). Blots were probed with antibodies specific for periplakin (Ppl; TD2), envoplakin (Evpl; CR5), or desmoplakin (Dp; 115F). As a control, 4% of cell lysate used in the pull-downs was loaded on gel (Extr). (D) Coimmunoprecipitation of kazrin C with P133. Cos-7 cells were transiently transfected with kazrin C-FLAG (K) together with empty vector (V) or P133-HA (P). Proteins were immunoprecipitated with anti-HA Y11 (HA IP), and immunoblots were probed with anti-FLAG mAb M2. As a control, 5% of cell lysate used in the immunoprecipitation was run in parallel (lysate). Positions of kazrin (K) and immunoglobulin light chain (Ig) are indicated.

Mentions: A BLAST search of the nucleotide sequence of the positive clone resulted in identification of four alternatively spliced isoforms encoded by the human gene, which we have named kazrin (Fig. 2). The kazrin gene is located on human chromosome 1 (band 1p36.21) and mouse chromosome 4 (band 4E1). Each isoform of human kazrin has one of four possible first exons, referred to as 1a, 1b, 1c, or 1d (Fig. 2 A). Exons 2 to 8 are spliced together and are preceded by one of the four alternative first exons (Fig. 2 A and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200312123/DC1).Exons 1a and 1b contain in-frame start codons resulting in proteins of 421 (kazrin A) and 414 (kazrin B) amino acids, respectively (Fig. S1 A). The coding sequence of kazrin splice forms containing 1c or 1d begins within exon 2, resulting in a protein of 327 amino acids. Thus, kazrin C and D have identical amino acid sequences and are referred to as kazrin C for simplicity. The region of kazrin identified in the yeast two-hybrid screen corresponds to amino acids 49 to 327 of kazrin C. BLAST searches against the mouse genome revealed the presence of sequences with homology to each of the alternative first exons 1a, 1b, 1c, 1d, and exons 2–8. Thus far, however, only sequences corresponding to kazrin a and b transcripts are present in mouse EST databases.


Kazrin, a novel periplakin-interacting protein associated with desmosomes and the keratinocyte plasma membrane.

Groot KR, Sevilla LM, Nishi K, DiColandrea T, Watt FM - J. Cell Biol. (2004)

Kazrin and its interaction with periplakin. (A) Intron-exon organization of the kazrin gene, drawn to scale. Arrows, translation initiation sites; diagonal lines, 320 kb gap; π, leucine zipper-like domain; black bar spanning exons 3 to 5, α-helical region; asterisk, putative NLS sequence. TGA, translation termination codon; AATAAA, polyadenylation signal. Sequence data available from GenBank/EMBL/DDBJ under accession no. AY505119, AY505120, AY505121, and AY505122, corresponding to kazrin isoform a, b, c, and d, respectively. (B) Secondary structure predictions of kazrin C, based on algorithms of Chou/Fasman and Garnier/Robson, and two-hybrid analysis of interaction between P133 and kazrin deletion constructs. Numbers correspond to amino acids of kazrin C. Interactions scored positive if yeast grew on selective medium. (C) Immunoblots of GST pull-downs from keratinocyte lysates with immobilized GST-kazrin A (KA), GST-kazrin B (KB), GST-kazrin C (KC), GST-kazrin C aa 49–327 (KCΔ), or GST alone (GST). Blots were probed with antibodies specific for periplakin (Ppl; TD2), envoplakin (Evpl; CR5), or desmoplakin (Dp; 115F). As a control, 4% of cell lysate used in the pull-downs was loaded on gel (Extr). (D) Coimmunoprecipitation of kazrin C with P133. Cos-7 cells were transiently transfected with kazrin C-FLAG (K) together with empty vector (V) or P133-HA (P). Proteins were immunoprecipitated with anti-HA Y11 (HA IP), and immunoblots were probed with anti-FLAG mAb M2. As a control, 5% of cell lysate used in the immunoprecipitation was run in parallel (lysate). Positions of kazrin (K) and immunoglobulin light chain (Ig) are indicated.
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fig2: Kazrin and its interaction with periplakin. (A) Intron-exon organization of the kazrin gene, drawn to scale. Arrows, translation initiation sites; diagonal lines, 320 kb gap; π, leucine zipper-like domain; black bar spanning exons 3 to 5, α-helical region; asterisk, putative NLS sequence. TGA, translation termination codon; AATAAA, polyadenylation signal. Sequence data available from GenBank/EMBL/DDBJ under accession no. AY505119, AY505120, AY505121, and AY505122, corresponding to kazrin isoform a, b, c, and d, respectively. (B) Secondary structure predictions of kazrin C, based on algorithms of Chou/Fasman and Garnier/Robson, and two-hybrid analysis of interaction between P133 and kazrin deletion constructs. Numbers correspond to amino acids of kazrin C. Interactions scored positive if yeast grew on selective medium. (C) Immunoblots of GST pull-downs from keratinocyte lysates with immobilized GST-kazrin A (KA), GST-kazrin B (KB), GST-kazrin C (KC), GST-kazrin C aa 49–327 (KCΔ), or GST alone (GST). Blots were probed with antibodies specific for periplakin (Ppl; TD2), envoplakin (Evpl; CR5), or desmoplakin (Dp; 115F). As a control, 4% of cell lysate used in the pull-downs was loaded on gel (Extr). (D) Coimmunoprecipitation of kazrin C with P133. Cos-7 cells were transiently transfected with kazrin C-FLAG (K) together with empty vector (V) or P133-HA (P). Proteins were immunoprecipitated with anti-HA Y11 (HA IP), and immunoblots were probed with anti-FLAG mAb M2. As a control, 5% of cell lysate used in the immunoprecipitation was run in parallel (lysate). Positions of kazrin (K) and immunoglobulin light chain (Ig) are indicated.
Mentions: A BLAST search of the nucleotide sequence of the positive clone resulted in identification of four alternatively spliced isoforms encoded by the human gene, which we have named kazrin (Fig. 2). The kazrin gene is located on human chromosome 1 (band 1p36.21) and mouse chromosome 4 (band 4E1). Each isoform of human kazrin has one of four possible first exons, referred to as 1a, 1b, 1c, or 1d (Fig. 2 A). Exons 2 to 8 are spliced together and are preceded by one of the four alternative first exons (Fig. 2 A and Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200312123/DC1).Exons 1a and 1b contain in-frame start codons resulting in proteins of 421 (kazrin A) and 414 (kazrin B) amino acids, respectively (Fig. S1 A). The coding sequence of kazrin splice forms containing 1c or 1d begins within exon 2, resulting in a protein of 327 amino acids. Thus, kazrin C and D have identical amino acid sequences and are referred to as kazrin C for simplicity. The region of kazrin identified in the yeast two-hybrid screen corresponds to amino acids 49 to 327 of kazrin C. BLAST searches against the mouse genome revealed the presence of sequences with homology to each of the alternative first exons 1a, 1b, 1c, 1d, and exons 2–8. Thus far, however, only sequences corresponding to kazrin a and b transcripts are present in mouse EST databases.

Bottom Line: Kazrin is expressed in all layers of stratified squamous epithelia; it becomes membrane associated in the suprabasal layers, coincident with up-regulation of periplakin, and is incorporated into the cornified envelope of cultured keratinocytes.On transfection, all three kazrin isoforms have similar subcellular distributions.We conclude that kazrin is a novel component of desmosomes that associates with periplakin.

View Article: PubMed Central - PubMed

Affiliation: Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.

ABSTRACT
Periplakin forms part of the scaffold onto which the epidermal cornified envelope is assembled. The NH2-terminal 133 amino acids mediate association with the plasma membrane and bind a novel protein, kazrin. Kazrin is highly conserved and lacks homology to any known protein. There are four alternatively spliced transcripts, encoding three proteins with different NH2 termini. Kazrin is expressed in all layers of stratified squamous epithelia; it becomes membrane associated in the suprabasal layers, coincident with up-regulation of periplakin, and is incorporated into the cornified envelope of cultured keratinocytes. Kazrin colocalizes with periplakin and desmoplakin at desmosomes and with periplakin at the interdesmosomal plasma membrane, but its subcellular distribution is independent of periplakin. On transfection, all three kazrin isoforms have similar subcellular distributions. We conclude that kazrin is a novel component of desmosomes that associates with periplakin.

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