Limits...
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.

Show MeSH
Phosphorylation of  myosin VI in vivo. (a) A431  cells were first labeled with  33P for 1 h and then used  unstimulated or were stimulated with EGF before immunoprecipitation of myosin VI  and ezrin. Phosphorylation  of myosin VI and ezrin increased after stimulation with  EGF. (b) Time course of  phosphorylation of myosin  VI and ezrin in A431 cells  after stimulation with EGF.  33P-labeled cells were stimulated before lysing the cells  for immunoprecipitation with  antibodies to myosin VI or  ezrin after 2, 5, or 15 min.  The amount of 33P incorporation into ezrin (▪) or myosin  VI (□) was quantified using a  phosphorImager and plotted  as a function of time. (c) Chymotryptic digest of 33P-phosphorylated myosin VI. The  33P-labeled myosin VI was  immunoprecipitated from  A431 cells and digested with  chymotrypsin before blotting  with the antibody to the head  domain of myosin VI, lanes 1  and 2. Lane 3 shows the autoradiogram of the blot shown  in lane 2. An asterisk marks  the bands recognized by the  ab to the head domain (H) in lane 2 and also labeled with  33P (lane 3), indicating that phosphorylation of myosin VI occurs  in the head domain.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132970&req=5

Figure 6: Phosphorylation of myosin VI in vivo. (a) A431 cells were first labeled with 33P for 1 h and then used unstimulated or were stimulated with EGF before immunoprecipitation of myosin VI and ezrin. Phosphorylation of myosin VI and ezrin increased after stimulation with EGF. (b) Time course of phosphorylation of myosin VI and ezrin in A431 cells after stimulation with EGF. 33P-labeled cells were stimulated before lysing the cells for immunoprecipitation with antibodies to myosin VI or ezrin after 2, 5, or 15 min. The amount of 33P incorporation into ezrin (▪) or myosin VI (□) was quantified using a phosphorImager and plotted as a function of time. (c) Chymotryptic digest of 33P-phosphorylated myosin VI. The 33P-labeled myosin VI was immunoprecipitated from A431 cells and digested with chymotrypsin before blotting with the antibody to the head domain of myosin VI, lanes 1 and 2. Lane 3 shows the autoradiogram of the blot shown in lane 2. An asterisk marks the bands recognized by the ab to the head domain (H) in lane 2 and also labeled with 33P (lane 3), indicating that phosphorylation of myosin VI occurs in the head domain.

Mentions: To investigate whether myosin VI like ezrin (Bretscher, 1989) was phosphorylated in vivo in A431 cells after stimulation with EGF we labeled cells with [33P]phosphate. After stimulation there was a clear increase in the level of phosphorylation of both myosin VI and ezrin (Fig. 6 a). We examined the time course of phosphorylation of myosin VI and ezrin after EGF stimulation and found that myosin VI was not as rapidly phosphorylated as ezrin but after 15 min the same increase in stimulation of phosphorylation was observed (Fig. 6 b). The slower increase in the rate of myosin VI phosphorylation was consistent with its slower recruitment into ruffles compared with phosphorylated ezrin (Fig. 5). Comparing the intensities of the radioactive bands on the autoradiogram with the Coomassie stained bands of myosin VI and ezrin on the gels suggested that the maximum level of phosphorylation of myosin VI was similar to that of ezrin.


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 of  myosin VI in vivo. (a) A431  cells were first labeled with  33P for 1 h and then used  unstimulated or were stimulated with EGF before immunoprecipitation of myosin VI  and ezrin. Phosphorylation  of myosin VI and ezrin increased after stimulation with  EGF. (b) Time course of  phosphorylation of myosin  VI and ezrin in A431 cells  after stimulation with EGF.  33P-labeled cells were stimulated before lysing the cells  for immunoprecipitation with  antibodies to myosin VI or  ezrin after 2, 5, or 15 min.  The amount of 33P incorporation into ezrin (▪) or myosin  VI (□) was quantified using a  phosphorImager and plotted  as a function of time. (c) Chymotryptic digest of 33P-phosphorylated myosin VI. The  33P-labeled myosin VI was  immunoprecipitated from  A431 cells and digested with  chymotrypsin before blotting  with the antibody to the head  domain of myosin VI, lanes 1  and 2. Lane 3 shows the autoradiogram of the blot shown  in lane 2. An asterisk marks  the bands recognized by the  ab to the head domain (H) in lane 2 and also labeled with  33P (lane 3), indicating that phosphorylation of myosin VI occurs  in the head domain.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Phosphorylation of myosin VI in vivo. (a) A431 cells were first labeled with 33P for 1 h and then used unstimulated or were stimulated with EGF before immunoprecipitation of myosin VI and ezrin. Phosphorylation of myosin VI and ezrin increased after stimulation with EGF. (b) Time course of phosphorylation of myosin VI and ezrin in A431 cells after stimulation with EGF. 33P-labeled cells were stimulated before lysing the cells for immunoprecipitation with antibodies to myosin VI or ezrin after 2, 5, or 15 min. The amount of 33P incorporation into ezrin (▪) or myosin VI (□) was quantified using a phosphorImager and plotted as a function of time. (c) Chymotryptic digest of 33P-phosphorylated myosin VI. The 33P-labeled myosin VI was immunoprecipitated from A431 cells and digested with chymotrypsin before blotting with the antibody to the head domain of myosin VI, lanes 1 and 2. Lane 3 shows the autoradiogram of the blot shown in lane 2. An asterisk marks the bands recognized by the ab to the head domain (H) in lane 2 and also labeled with 33P (lane 3), indicating that phosphorylation of myosin VI occurs in the head domain.
Mentions: To investigate whether myosin VI like ezrin (Bretscher, 1989) was phosphorylated in vivo in A431 cells after stimulation with EGF we labeled cells with [33P]phosphate. After stimulation there was a clear increase in the level of phosphorylation of both myosin VI and ezrin (Fig. 6 a). We examined the time course of phosphorylation of myosin VI and ezrin after EGF stimulation and found that myosin VI was not as rapidly phosphorylated as ezrin but after 15 min the same increase in stimulation of phosphorylation was observed (Fig. 6 b). The slower increase in the rate of myosin VI phosphorylation was consistent with its slower recruitment into ruffles compared with phosphorylated ezrin (Fig. 5). Comparing the intensities of the radioactive bands on the autoradiogram with the Coomassie stained bands of myosin VI and ezrin on the gels suggested that the maximum level of phosphorylation of myosin VI was similar to that of ezrin.

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