Limits...
The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

Leung CL, Sun D, Liem RK - J. Cell Biol. (1999)

Bottom Line: Kouklis, D.W.Fuchs. 1996.Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Show MeSH

Related in: MedlinePlus

Specific interactions of mBPAG-C2 with peripherin in overlay binding  assays. (A) Coomassie brilliant blue staining of purified  NF-L (lane 1), NF-M (lane  2), NF-H (lane 3), peripherin  (lane 4), and FLAG-mBPAG-C2-His (lane 5). (B)  Equal amounts of NF-L (lane  1), NF-M (lane 2), NF-H  (lane 3), and peripherin (lane  4) were subjected to SDS-PAGE and electrotransferred onto a nylon membrane. The membrane  containing the nIF proteins  was incubated with FLAG-mBPAG-C2-His and the  bound FLAG-BPAG-C2-His  was visualized by immunoblotting with monoclonal anti- FLAG M2 antibody. Lane 5  is a Western blot of FLAG-mBPAG-C2-His detected by  the anti-FLAG antibody. (C)  Peripherin (P), NF-L/NF-M  (L/M), and NF-L/NF-H (L/H) were polymerized in vitro, dotted  onto a nitrocellulose membrane, and incubated with FLAG-mBPAG-C2-His. Bound FLAG-mBPAG-C2-His was visualized  with the anti-FLAG M2 antibody and quantified as described.  Data shown are averages of triplicate determinations.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132913&req=5

Figure 5: Specific interactions of mBPAG-C2 with peripherin in overlay binding assays. (A) Coomassie brilliant blue staining of purified NF-L (lane 1), NF-M (lane 2), NF-H (lane 3), peripherin (lane 4), and FLAG-mBPAG-C2-His (lane 5). (B) Equal amounts of NF-L (lane 1), NF-M (lane 2), NF-H (lane 3), and peripherin (lane 4) were subjected to SDS-PAGE and electrotransferred onto a nylon membrane. The membrane containing the nIF proteins was incubated with FLAG-mBPAG-C2-His and the bound FLAG-BPAG-C2-His was visualized by immunoblotting with monoclonal anti- FLAG M2 antibody. Lane 5 is a Western blot of FLAG-mBPAG-C2-His detected by the anti-FLAG antibody. (C) Peripherin (P), NF-L/NF-M (L/M), and NF-L/NF-H (L/H) were polymerized in vitro, dotted onto a nitrocellulose membrane, and incubated with FLAG-mBPAG-C2-His. Bound FLAG-mBPAG-C2-His was visualized with the anti-FLAG M2 antibody and quantified as described. Data shown are averages of triplicate determinations.

Mentions: To confirm the specificity of the interaction between mBPAG-C2 and peripherin, we also used in vitro overlay binding assays. Peripherin was chosen, instead of α-internexin, because its in vivo expression pattern resembles that of BPAG1-n; therefore, their interaction is of more physiological significance (Parysek and Goldman, 1988; Troy et al., 1990a; Dowling et al., 1997). Recent studies on the interactions of plectin and desmoplakin with IF proteins demonstrated that the association between these proteins can be monitored by overlay binding assays (Nikolic et al., 1996; Meng et al., 1997). Hence, we examined the interactions between mBPAG-C2 and IF proteins by similar strategies. Bacterially expressed IF proteins and mBPAG-C2 were successfully purified by column chromatography (Fig. 5 A). Purified IF proteins were resolved by SDS-PAGE and transferred onto a nylon membrane. After the removal of SDS by washing in PBS (0.05% Tween 20 and 5% BSA), the membrane was incubated with mBPAG-C2 protein. The bound mBPAG-C2 was detected by an anti-FLAG mAb. As illustrated in Fig. 5 B, mBPAG-C2 associated with peripherin protein, but not with the NFTPs.


The intermediate filament protein peripherin is the specific interaction partner of mouse BPAG1-n (dystonin) in neurons.

Leung CL, Sun D, Liem RK - J. Cell Biol. (1999)

Specific interactions of mBPAG-C2 with peripherin in overlay binding  assays. (A) Coomassie brilliant blue staining of purified  NF-L (lane 1), NF-M (lane  2), NF-H (lane 3), peripherin  (lane 4), and FLAG-mBPAG-C2-His (lane 5). (B)  Equal amounts of NF-L (lane  1), NF-M (lane 2), NF-H  (lane 3), and peripherin (lane  4) were subjected to SDS-PAGE and electrotransferred onto a nylon membrane. The membrane  containing the nIF proteins  was incubated with FLAG-mBPAG-C2-His and the  bound FLAG-BPAG-C2-His  was visualized by immunoblotting with monoclonal anti- FLAG M2 antibody. Lane 5  is a Western blot of FLAG-mBPAG-C2-His detected by  the anti-FLAG antibody. (C)  Peripherin (P), NF-L/NF-M  (L/M), and NF-L/NF-H (L/H) were polymerized in vitro, dotted  onto a nitrocellulose membrane, and incubated with FLAG-mBPAG-C2-His. Bound FLAG-mBPAG-C2-His was visualized  with the anti-FLAG M2 antibody and quantified as described.  Data shown are averages of triplicate determinations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132913&req=5

Figure 5: Specific interactions of mBPAG-C2 with peripherin in overlay binding assays. (A) Coomassie brilliant blue staining of purified NF-L (lane 1), NF-M (lane 2), NF-H (lane 3), peripherin (lane 4), and FLAG-mBPAG-C2-His (lane 5). (B) Equal amounts of NF-L (lane 1), NF-M (lane 2), NF-H (lane 3), and peripherin (lane 4) were subjected to SDS-PAGE and electrotransferred onto a nylon membrane. The membrane containing the nIF proteins was incubated with FLAG-mBPAG-C2-His and the bound FLAG-BPAG-C2-His was visualized by immunoblotting with monoclonal anti- FLAG M2 antibody. Lane 5 is a Western blot of FLAG-mBPAG-C2-His detected by the anti-FLAG antibody. (C) Peripherin (P), NF-L/NF-M (L/M), and NF-L/NF-H (L/H) were polymerized in vitro, dotted onto a nitrocellulose membrane, and incubated with FLAG-mBPAG-C2-His. Bound FLAG-mBPAG-C2-His was visualized with the anti-FLAG M2 antibody and quantified as described. Data shown are averages of triplicate determinations.
Mentions: To confirm the specificity of the interaction between mBPAG-C2 and peripherin, we also used in vitro overlay binding assays. Peripherin was chosen, instead of α-internexin, because its in vivo expression pattern resembles that of BPAG1-n; therefore, their interaction is of more physiological significance (Parysek and Goldman, 1988; Troy et al., 1990a; Dowling et al., 1997). Recent studies on the interactions of plectin and desmoplakin with IF proteins demonstrated that the association between these proteins can be monitored by overlay binding assays (Nikolic et al., 1996; Meng et al., 1997). Hence, we examined the interactions between mBPAG-C2 and IF proteins by similar strategies. Bacterially expressed IF proteins and mBPAG-C2 were successfully purified by column chromatography (Fig. 5 A). Purified IF proteins were resolved by SDS-PAGE and transferred onto a nylon membrane. After the removal of SDS by washing in PBS (0.05% Tween 20 and 5% BSA), the membrane was incubated with mBPAG-C2 protein. The bound mBPAG-C2 was detected by an anti-FLAG mAb. As illustrated in Fig. 5 B, mBPAG-C2 associated with peripherin protein, but not with the NFTPs.

Bottom Line: Kouklis, D.W.Fuchs. 1996.Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biophysics, Columbia University College of Physicians and Surgeons, New York 10032, USA.

ABSTRACT
The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH2 terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655-665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.

Show MeSH
Related in: MedlinePlus