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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 protein expression and cellular localization. (A) Immunoblot of protein lysates from A431, EJ/28, and human keratinocytes (HK) with anti-kazrin (LS4) or anti-actin (loading control). The 37-kD band corresponds to kazrin C (C); the 47-kD doublet corresponds to kazrin A and B (A/B). Molecular mass markers (kD) are indicated. White line indicates that intervening lanes have been spliced out. (B) CE incorporation of periplakin and kazrin. Keratinocytes were incubated in the absence (−) or presence (T) of Triton X-100, cystamine (Cy), or cystamine and Triton X-100 (Cy/T). Immunoblots were probed with antibodies to kazrin (46 kD isoforms A/B are shown; K) and periplakin (P). (C–I) Immunofluorescence staining of human interfollicular epidermis (C and D) and hair follicles (E–I) with antibodies specific for kazrin (K; green in C, E, and G–I), periplakin (PPL; D, green; and I, red), or desmoplakin (DP; F–H, red). (C and D) Nuclei were labeled with TO-PRO-3 (blue). (G) Boxed area is enlarged in H. Bars: (C–G) 50 μm; (H and I) 6 μm. (J–M) Immunogold EM of human breast epidermis with anti-desmoplakin (J) and anti-kazrin (K–M). Arrows indicate 10-nm gold particles localized at desmosomes (J–L) or in nucleus (M). Bar: (J) 200 nm; (K) 330 nm; (L) 120 nm; (M) 487 nm.
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fig4: Kazrin protein expression and cellular localization. (A) Immunoblot of protein lysates from A431, EJ/28, and human keratinocytes (HK) with anti-kazrin (LS4) or anti-actin (loading control). The 37-kD band corresponds to kazrin C (C); the 47-kD doublet corresponds to kazrin A and B (A/B). Molecular mass markers (kD) are indicated. White line indicates that intervening lanes have been spliced out. (B) CE incorporation of periplakin and kazrin. Keratinocytes were incubated in the absence (−) or presence (T) of Triton X-100, cystamine (Cy), or cystamine and Triton X-100 (Cy/T). Immunoblots were probed with antibodies to kazrin (46 kD isoforms A/B are shown; K) and periplakin (P). (C–I) Immunofluorescence staining of human interfollicular epidermis (C and D) and hair follicles (E–I) with antibodies specific for kazrin (K; green in C, E, and G–I), periplakin (PPL; D, green; and I, red), or desmoplakin (DP; F–H, red). (C and D) Nuclei were labeled with TO-PRO-3 (blue). (G) Boxed area is enlarged in H. Bars: (C–G) 50 μm; (H and I) 6 μm. (J–M) Immunogold EM of human breast epidermis with anti-desmoplakin (J) and anti-kazrin (K–M). Arrows indicate 10-nm gold particles localized at desmosomes (J–L) or in nucleus (M). Bar: (J) 200 nm; (K) 330 nm; (L) 120 nm; (M) 487 nm.

Mentions: Affinity-purified rabbit anti-kazrin (LS4) recognized bands of ∼47 and 37 kD on Western blots of cell lysates (Fig. 4 A). These bands correspond to the predicted molecular masses of kazrin A (46.7 kD), kazrin B (46 kD), and kazrin C (37 kD). A431 cells express kazrin b but not kazrin a transcript (Fig. 3 A); nevertheless the 47-kD protein band from A431 lysates appeared as a doublet, suggesting kazrin may undergo posttranslational modification (Fig. 4 A). Although A431, EJ/28, and human keratinocytes expressed similar levels of kazrin d transcript (Fig. 3 A), the 37-kD form of kazrin protein was relatively less abundant in human keratinocytes, an indication of differential regulation at the translational or posttranslational level.


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 protein expression and cellular localization. (A) Immunoblot of protein lysates from A431, EJ/28, and human keratinocytes (HK) with anti-kazrin (LS4) or anti-actin (loading control). The 37-kD band corresponds to kazrin C (C); the 47-kD doublet corresponds to kazrin A and B (A/B). Molecular mass markers (kD) are indicated. White line indicates that intervening lanes have been spliced out. (B) CE incorporation of periplakin and kazrin. Keratinocytes were incubated in the absence (−) or presence (T) of Triton X-100, cystamine (Cy), or cystamine and Triton X-100 (Cy/T). Immunoblots were probed with antibodies to kazrin (46 kD isoforms A/B are shown; K) and periplakin (P). (C–I) Immunofluorescence staining of human interfollicular epidermis (C and D) and hair follicles (E–I) with antibodies specific for kazrin (K; green in C, E, and G–I), periplakin (PPL; D, green; and I, red), or desmoplakin (DP; F–H, red). (C and D) Nuclei were labeled with TO-PRO-3 (blue). (G) Boxed area is enlarged in H. Bars: (C–G) 50 μm; (H and I) 6 μm. (J–M) Immunogold EM of human breast epidermis with anti-desmoplakin (J) and anti-kazrin (K–M). Arrows indicate 10-nm gold particles localized at desmosomes (J–L) or in nucleus (M). Bar: (J) 200 nm; (K) 330 nm; (L) 120 nm; (M) 487 nm.
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fig4: Kazrin protein expression and cellular localization. (A) Immunoblot of protein lysates from A431, EJ/28, and human keratinocytes (HK) with anti-kazrin (LS4) or anti-actin (loading control). The 37-kD band corresponds to kazrin C (C); the 47-kD doublet corresponds to kazrin A and B (A/B). Molecular mass markers (kD) are indicated. White line indicates that intervening lanes have been spliced out. (B) CE incorporation of periplakin and kazrin. Keratinocytes were incubated in the absence (−) or presence (T) of Triton X-100, cystamine (Cy), or cystamine and Triton X-100 (Cy/T). Immunoblots were probed with antibodies to kazrin (46 kD isoforms A/B are shown; K) and periplakin (P). (C–I) Immunofluorescence staining of human interfollicular epidermis (C and D) and hair follicles (E–I) with antibodies specific for kazrin (K; green in C, E, and G–I), periplakin (PPL; D, green; and I, red), or desmoplakin (DP; F–H, red). (C and D) Nuclei were labeled with TO-PRO-3 (blue). (G) Boxed area is enlarged in H. Bars: (C–G) 50 μm; (H and I) 6 μm. (J–M) Immunogold EM of human breast epidermis with anti-desmoplakin (J) and anti-kazrin (K–M). Arrows indicate 10-nm gold particles localized at desmosomes (J–L) or in nucleus (M). Bar: (J) 200 nm; (K) 330 nm; (L) 120 nm; (M) 487 nm.
Mentions: Affinity-purified rabbit anti-kazrin (LS4) recognized bands of ∼47 and 37 kD on Western blots of cell lysates (Fig. 4 A). These bands correspond to the predicted molecular masses of kazrin A (46.7 kD), kazrin B (46 kD), and kazrin C (37 kD). A431 cells express kazrin b but not kazrin a transcript (Fig. 3 A); nevertheless the 47-kD protein band from A431 lysates appeared as a doublet, suggesting kazrin may undergo posttranslational modification (Fig. 4 A). Although A431, EJ/28, and human keratinocytes expressed similar levels of kazrin d transcript (Fig. 3 A), the 37-kD form of kazrin protein was relatively less abundant in human keratinocytes, an indication of differential regulation at the translational or posttranslational level.

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.

Show MeSH
Related in: MedlinePlus