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The localization of myosin VI at the golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation.

Buss F, Kendrick-Jones J, Lionne C, Knight AE, Côté GP, Paul Luzio J - J. Cell Biol. (1998)

Bottom Line: It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool.In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain.These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom. fb1@mole.bio.cam.ac.uk

ABSTRACT
Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

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Amino acid sequence of the chicken brush border myosin VI. (a) Schematic representation of the domain organization  and special features in the sequence. (b) Alignment of the  chicken brush border myosin VI sequence (top) with porcine myosin VI (bottom). Dots indicate identical amino acids, whereas  capital letters indicate a different amino acid. Features are highlighted as follows (starting from the NH2-terminus): box surrounded by dashed line, 22 amino acid insert in the head domain;  *threonine406 a possible phosphorylation site for a specific heavy  chain kinase; box surrounded by solid line with sequence dashed  underlined, spacer region COOH-terminal of the motor domain;  dashed underlined, IQ motif; box surrounded by solid line, region  of predicted coiled coil; bold letters and underlined within this  coiled coil region are charged repeats; shaded box, 23-aa insert in  the myosin VI tail.
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Figure 1: Amino acid sequence of the chicken brush border myosin VI. (a) Schematic representation of the domain organization and special features in the sequence. (b) Alignment of the chicken brush border myosin VI sequence (top) with porcine myosin VI (bottom). Dots indicate identical amino acids, whereas capital letters indicate a different amino acid. Features are highlighted as follows (starting from the NH2-terminus): box surrounded by dashed line, 22 amino acid insert in the head domain; *threonine406 a possible phosphorylation site for a specific heavy chain kinase; box surrounded by solid line with sequence dashed underlined, spacer region COOH-terminal of the motor domain; dashed underlined, IQ motif; box surrounded by solid line, region of predicted coiled coil; bold letters and underlined within this coiled coil region are charged repeats; shaded box, 23-aa insert in the myosin VI tail.

Mentions: A full-length cDNA encoding a chicken brush border myosin VI was isolated and sequenced. The amino acid sequence (Fig. 1) is 90% identical to that of pig myosin VI (Hasson and Mooseker, 1994) and 60% identical to Drosophila 95F myosin VI (Kellerman and Miller, 1992). The chicken myosin VI has a predicted molecular mass of 147.7 kD. The amount of myosin VI present in tissue culture cells such as human epidermoid carcinoma A431 cells was estimated as 0.15% of the total protein present. All of the myosin VIs so far sequenced have a number of unique features compared with other myosins (Hasson and Mooseker, 1994). Within the motor domain there is a small insert of 22 residues (276–297). After the motor domain there is a ∼53 residue spacer region with no known structural motifs (maybe it binds an unusual light chain subunit) followed by a single IQ motif which is predicted to bind calmodulin. Directly after the IQ motif there is a region predicted to form an α-helical coiled coil, both by the algorithm of Andrei Lupas (Lupas et al., 1991) and by the Paircoil method (Berger et al., 1995). This coiled coil seems to fall into two blocks at residues 846–887 and 921– 1002 (see Fig. 1). However, overlapping with the second block is a region containing several repeats of charged aa (916–981). These repeats typically consist of four basic residues followed by four acidic residues, e.g., RKRREEEE. This region is well conserved among the vertebrate myosin VI sequences and seems most unlikely to form a stable coiled coil structure. Secondary structure predictions using the nnpredict method (Kneller et al., 1990) amongst others suggest that most of these charged residues are in an alpha helix. Searching protein sequence databases for a similar sequence repeat using the PatternFind server (http://ulrec3.unil.ch/software/software.html) found very few proteins with similar repeats. It seems most likely that this structure would form a helix that is completely exposed to the aqueous environment, with alternating bands of positively and negatively charged residues on its surface which may stabilize the helix. Immediately after the end of this region there is an insertion of 23 aa in the chicken myosin VI sequence compared with the porcine myosin VI sequence (Hasson and Mooseker, 1994), which suggests that the chicken myosin VI gene maybe differentially spliced in the tail. Preliminary PCR analysis indicates that the transcript with the 23-aa insert is the major species in the chicken brush border cDNA library but both isoforms with and without the insert are present in various ratios in several vertebrate libraries screened to date (data not shown).


The localization of myosin VI at the golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation.

Buss F, Kendrick-Jones J, Lionne C, Knight AE, Côté GP, Paul Luzio J - J. Cell Biol. (1998)

Amino acid sequence of the chicken brush border myosin VI. (a) Schematic representation of the domain organization  and special features in the sequence. (b) Alignment of the  chicken brush border myosin VI sequence (top) with porcine myosin VI (bottom). Dots indicate identical amino acids, whereas  capital letters indicate a different amino acid. Features are highlighted as follows (starting from the NH2-terminus): box surrounded by dashed line, 22 amino acid insert in the head domain;  *threonine406 a possible phosphorylation site for a specific heavy  chain kinase; box surrounded by solid line with sequence dashed  underlined, spacer region COOH-terminal of the motor domain;  dashed underlined, IQ motif; box surrounded by solid line, region  of predicted coiled coil; bold letters and underlined within this  coiled coil region are charged repeats; shaded box, 23-aa insert in  the myosin VI tail.
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Related In: Results  -  Collection

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Figure 1: Amino acid sequence of the chicken brush border myosin VI. (a) Schematic representation of the domain organization and special features in the sequence. (b) Alignment of the chicken brush border myosin VI sequence (top) with porcine myosin VI (bottom). Dots indicate identical amino acids, whereas capital letters indicate a different amino acid. Features are highlighted as follows (starting from the NH2-terminus): box surrounded by dashed line, 22 amino acid insert in the head domain; *threonine406 a possible phosphorylation site for a specific heavy chain kinase; box surrounded by solid line with sequence dashed underlined, spacer region COOH-terminal of the motor domain; dashed underlined, IQ motif; box surrounded by solid line, region of predicted coiled coil; bold letters and underlined within this coiled coil region are charged repeats; shaded box, 23-aa insert in the myosin VI tail.
Mentions: A full-length cDNA encoding a chicken brush border myosin VI was isolated and sequenced. The amino acid sequence (Fig. 1) is 90% identical to that of pig myosin VI (Hasson and Mooseker, 1994) and 60% identical to Drosophila 95F myosin VI (Kellerman and Miller, 1992). The chicken myosin VI has a predicted molecular mass of 147.7 kD. The amount of myosin VI present in tissue culture cells such as human epidermoid carcinoma A431 cells was estimated as 0.15% of the total protein present. All of the myosin VIs so far sequenced have a number of unique features compared with other myosins (Hasson and Mooseker, 1994). Within the motor domain there is a small insert of 22 residues (276–297). After the motor domain there is a ∼53 residue spacer region with no known structural motifs (maybe it binds an unusual light chain subunit) followed by a single IQ motif which is predicted to bind calmodulin. Directly after the IQ motif there is a region predicted to form an α-helical coiled coil, both by the algorithm of Andrei Lupas (Lupas et al., 1991) and by the Paircoil method (Berger et al., 1995). This coiled coil seems to fall into two blocks at residues 846–887 and 921– 1002 (see Fig. 1). However, overlapping with the second block is a region containing several repeats of charged aa (916–981). These repeats typically consist of four basic residues followed by four acidic residues, e.g., RKRREEEE. This region is well conserved among the vertebrate myosin VI sequences and seems most unlikely to form a stable coiled coil structure. Secondary structure predictions using the nnpredict method (Kneller et al., 1990) amongst others suggest that most of these charged residues are in an alpha helix. Searching protein sequence databases for a similar sequence repeat using the PatternFind server (http://ulrec3.unil.ch/software/software.html) found very few proteins with similar repeats. It seems most likely that this structure would form a helix that is completely exposed to the aqueous environment, with alternating bands of positively and negatively charged residues on its surface which may stabilize the helix. Immediately after the end of this region there is an insertion of 23 aa in the chicken myosin VI sequence compared with the porcine myosin VI sequence (Hasson and Mooseker, 1994), which suggests that the chicken myosin VI gene maybe differentially spliced in the tail. Preliminary PCR analysis indicates that the transcript with the 23-aa insert is the major species in the chicken brush border cDNA library but both isoforms with and without the insert are present in various ratios in several vertebrate libraries screened to date (data not shown).

Bottom Line: It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool.In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain.These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QR, United Kingdom. fb1@mole.bio.cam.ac.uk

ABSTRACT
Myosin VI is an unconventional myosin that may play a role in vesicular membrane traffic through actin rich regions of the cytoplasm in eukaryotic cells. In this study we have cloned and sequenced a cDNA encoding a chicken intestinal brush border myosin VI. Polyclonal antisera were raised to bacterially expressed fragments of this myosin VI. The affinity purified antibodies were highly specific for myosin VI by immunoblotting and immunoprecipitation and were used to study the localization of the protein by immunofluorescence and immunoelectron microscopy. It was found that in NRK and A431 cells, myosin VI was associated with both the Golgi complex and the leading, ruffling edge of the cell as well as being present in a cytosolic pool. In A431 cells in which cell surface ruffling was stimulated by EGF, myosin VI was phosphorylated and recruited into the newly formed ruffles along with ezrin and myosin V. In vitro experiments suggested that a p21-activated kinase (PAK) might be the kinase responsible for phosphorylation in the motor domain. These results strongly support a role for myosin VI in membrane traffic on secretory and endocytic pathways.

Show MeSH