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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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Phosphorylation in vitro by PAK. (a) Myosin VI and  myosin I (myr 2) were immunoprecipitated from A431 cells and  then incubated with recombinant GST-PAK and γ-[32P]ATP for  30 min. Myosin VI (148 kD) but not myosin I (myr 2, 118 kD)  was clearly phosphorylated by PAK, which also autophosphorylates and results in a band at 105 kD (b). Myosin VI head fragment (aa 308–631) was expressed in E. coli as a GST-fusion protein, purified and incubated with recombinant GST-PAK and  γ-[32P]ATP for 30 min. As controls bacterially expressed GST or  the motor domain (subfragment 1) of smooth muscle myosin II  (MII S1) were incubated with PAK. The major heavy chain fragment in the smooth muscle myosin subfragment 1 runs at 90 kD  with a minor heavy chain fragment at 70 kD. Lane 1 shows the  Coomassie stained gel and lane 2 the corresponding autoradiogram of the same gel.
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Figure 7: Phosphorylation in vitro by PAK. (a) Myosin VI and myosin I (myr 2) were immunoprecipitated from A431 cells and then incubated with recombinant GST-PAK and γ-[32P]ATP for 30 min. Myosin VI (148 kD) but not myosin I (myr 2, 118 kD) was clearly phosphorylated by PAK, which also autophosphorylates and results in a band at 105 kD (b). Myosin VI head fragment (aa 308–631) was expressed in E. coli as a GST-fusion protein, purified and incubated with recombinant GST-PAK and γ-[32P]ATP for 30 min. As controls bacterially expressed GST or the motor domain (subfragment 1) of smooth muscle myosin II (MII S1) were incubated with PAK. The major heavy chain fragment in the smooth muscle myosin subfragment 1 runs at 90 kD with a minor heavy chain fragment at 70 kD. Lane 1 shows the Coomassie stained gel and lane 2 the corresponding autoradiogram of the same gel.

Mentions: The motor domain (heavy chain) of amoeboid myosin Is is phosphorylated by specific myosin I heavy chain kinases (MIHCK). Since these MIHCKs have recently been shown to be homologous to the mammalian PAK (Brzeska and Korn, 1996; Sells and Chernoff, 1997) we tested whether the myosin VI from A431 cells could be phosphorylated by PAK. Myosin VI and a mammalian myosin I (myr 2; Ruppert et al., 1995), as a control, were therefore immunoprecipitated from A431 cells and incubated with activated recombinant GST-PAK in the presence of γ-[32P]ATP. PAK clearly phosphorylates the myosin VI (MVI) but not the myosin I (myr 2; Fig. 7 a). The PAK was also able to phosphorylate, in vitro, a recombinant myosin VI head fragment (aa 308–631) bacterially expressed as a GST fusion protein. This fragment contains the predicted MIHCK/ PAK phosphorylation site at threonine residue 406. Under the same conditions, bacterially expressed GST and purified subfragment 1 from smooth muscle myosin II were not phosphorylated (Fig. 7 b). Interestingly the regulatory light chain of smooth muscle myosin II was phosphorylated under these conditions.


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)

Phosphorylation in vitro by PAK. (a) Myosin VI and  myosin I (myr 2) were immunoprecipitated from A431 cells and  then incubated with recombinant GST-PAK and γ-[32P]ATP for  30 min. Myosin VI (148 kD) but not myosin I (myr 2, 118 kD)  was clearly phosphorylated by PAK, which also autophosphorylates and results in a band at 105 kD (b). Myosin VI head fragment (aa 308–631) was expressed in E. coli as a GST-fusion protein, purified and incubated with recombinant GST-PAK and  γ-[32P]ATP for 30 min. As controls bacterially expressed GST or  the motor domain (subfragment 1) of smooth muscle myosin II  (MII S1) were incubated with PAK. The major heavy chain fragment in the smooth muscle myosin subfragment 1 runs at 90 kD  with a minor heavy chain fragment at 70 kD. Lane 1 shows the  Coomassie stained gel and lane 2 the corresponding autoradiogram of the same gel.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132970&req=5

Figure 7: Phosphorylation in vitro by PAK. (a) Myosin VI and myosin I (myr 2) were immunoprecipitated from A431 cells and then incubated with recombinant GST-PAK and γ-[32P]ATP for 30 min. Myosin VI (148 kD) but not myosin I (myr 2, 118 kD) was clearly phosphorylated by PAK, which also autophosphorylates and results in a band at 105 kD (b). Myosin VI head fragment (aa 308–631) was expressed in E. coli as a GST-fusion protein, purified and incubated with recombinant GST-PAK and γ-[32P]ATP for 30 min. As controls bacterially expressed GST or the motor domain (subfragment 1) of smooth muscle myosin II (MII S1) were incubated with PAK. The major heavy chain fragment in the smooth muscle myosin subfragment 1 runs at 90 kD with a minor heavy chain fragment at 70 kD. Lane 1 shows the Coomassie stained gel and lane 2 the corresponding autoradiogram of the same gel.
Mentions: The motor domain (heavy chain) of amoeboid myosin Is is phosphorylated by specific myosin I heavy chain kinases (MIHCK). Since these MIHCKs have recently been shown to be homologous to the mammalian PAK (Brzeska and Korn, 1996; Sells and Chernoff, 1997) we tested whether the myosin VI from A431 cells could be phosphorylated by PAK. Myosin VI and a mammalian myosin I (myr 2; Ruppert et al., 1995), as a control, were therefore immunoprecipitated from A431 cells and incubated with activated recombinant GST-PAK in the presence of γ-[32P]ATP. PAK clearly phosphorylates the myosin VI (MVI) but not the myosin I (myr 2; Fig. 7 a). The PAK was also able to phosphorylate, in vitro, a recombinant myosin VI head fragment (aa 308–631) bacterially expressed as a GST fusion protein. This fragment contains the predicted MIHCK/ PAK phosphorylation site at threonine residue 406. Under the same conditions, bacterially expressed GST and purified subfragment 1 from smooth muscle myosin II were not phosphorylated (Fig. 7 b). Interestingly the regulatory light chain of smooth muscle myosin II was phosphorylated under these conditions.

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
Related in: MedlinePlus