Limits...
Annexin 2 has a dual role as regulator and effector of v-Src in cell transformation.

Hayes MJ, Moss SE - J. Biol. Chem. (2009)

Bottom Line: Here, we report that this is dependent on annexin 2, a v-Src substrate and calcium-dependent regulator of actin dynamics.In cells depleted of annexin 2 by small interfering RNA, v-Src becomes activated at the permissive temperature but does not target to the plasma membrane or to perinuclear vesicles, and cell transformation does not occur.Our findings reveal a dual role for annexin 2, first as a regulator of v-Src trafficking and targeting and second as a v-Src effector in the reorganization of actin.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, University College London Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, United Kingdom.

ABSTRACT
Cell transformation by v-Src involves rearrangement of the actin cytoskeleton, disassembly of focal adhesions, and the development of anchorage-independent growth. Here, we report that this is dependent on annexin 2, a v-Src substrate and calcium-dependent regulator of actin dynamics. Using a thermoactivatable mutant of v-Src, we show that at the permissive temperature, annexin 2 becomes phosphorylated and colocalizes with activated v-Src and focal adhesion kinase both at the plasma membrane and in a Rab11-positive compartment of the endosomal pathway. In cells depleted of annexin 2 by small interfering RNA, v-Src becomes activated at the permissive temperature but does not target to the plasma membrane or to perinuclear vesicles, and cell transformation does not occur. Our findings reveal a dual role for annexin 2, first as a regulator of v-Src trafficking and targeting and second as a v-Src effector in the reorganization of actin.

Show MeSH

Related in: MedlinePlus

Membrane targeting of activated Src requires annexin 2. A, R1LA29 cells were depleted of annexin 2 by siRNA treatment for 4 days at 41 °C. Cultures were then moved to 35 °C for the times indicated prior to fixation and immunostaining for annexin 2 (anx 2), Src-pY416, and F-actin. The top panel shows confocal sections at the base of cells grown at 41 °C, with both annexin 2-positive and annexin 2-negative cells in the field of view. Annexin 2 is present throughout the cytoplasm, the actin cytoskeleton is dominated by stress fibers, and Src-pY416 is undetectable. After 4 h at 35 °C, annexin 2, actin, and Src-pY416 appear at the actin-rich cortex and in a perinuclear vesicular domain (pink arrowheads), whereas there is no appearance of Src-pY416 at these sites and no change in cell shape in annexin 2-depleted cells. After 24 h, Src-pY416 is clearly enriched at the focal adhesions (white arrowheads) in annexin 2-depleted cells, but there is no morphological transformation. Insets show diagrammatic representations of the confocal plane. Scale bars, 25 μm. B, whole cell extracts were prepared from R1LA29 cells following a 4-day culture at 41 °C in the presence of control or annexin 2 siRNA and from cells that had been switched to the permissive temperature for the times indicated and immunoblotted as described in the legend to Fig. 1B. The right panel shows extracts from the control siRNA-treated cells at the restrictive temperature and after 36 h at the permissive temperature. There is an increase in phosphorylation of Src on phospho-Tyr416 and of paxillin on phospho-Tyr118, but phosphorylation on FAK at phospho-Tyr576 is impaired. C, densitometric analysis of Src-pY416 Western blots in control and annexin 2-depleted cells shows that the band intensity, indicative of Src activation, increases in a similar manner under both experimental conditions. KD, knockdown. D, a densitometric analysis shows that annexin 2 levels are maintained at least 50% below the control (Ctrl; when normalized to tubulin) for the duration of the experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2665074&req=5

fig2: Membrane targeting of activated Src requires annexin 2. A, R1LA29 cells were depleted of annexin 2 by siRNA treatment for 4 days at 41 °C. Cultures were then moved to 35 °C for the times indicated prior to fixation and immunostaining for annexin 2 (anx 2), Src-pY416, and F-actin. The top panel shows confocal sections at the base of cells grown at 41 °C, with both annexin 2-positive and annexin 2-negative cells in the field of view. Annexin 2 is present throughout the cytoplasm, the actin cytoskeleton is dominated by stress fibers, and Src-pY416 is undetectable. After 4 h at 35 °C, annexin 2, actin, and Src-pY416 appear at the actin-rich cortex and in a perinuclear vesicular domain (pink arrowheads), whereas there is no appearance of Src-pY416 at these sites and no change in cell shape in annexin 2-depleted cells. After 24 h, Src-pY416 is clearly enriched at the focal adhesions (white arrowheads) in annexin 2-depleted cells, but there is no morphological transformation. Insets show diagrammatic representations of the confocal plane. Scale bars, 25 μm. B, whole cell extracts were prepared from R1LA29 cells following a 4-day culture at 41 °C in the presence of control or annexin 2 siRNA and from cells that had been switched to the permissive temperature for the times indicated and immunoblotted as described in the legend to Fig. 1B. The right panel shows extracts from the control siRNA-treated cells at the restrictive temperature and after 36 h at the permissive temperature. There is an increase in phosphorylation of Src on phospho-Tyr416 and of paxillin on phospho-Tyr118, but phosphorylation on FAK at phospho-Tyr576 is impaired. C, densitometric analysis of Src-pY416 Western blots in control and annexin 2-depleted cells shows that the band intensity, indicative of Src activation, increases in a similar manner under both experimental conditions. KD, knockdown. D, a densitometric analysis shows that annexin 2 levels are maintained at least 50% below the control (Ctrl; when normalized to tubulin) for the duration of the experiment.

Mentions: Annexin 2 Is Required for Plasma Membrane Targeting of v-Src—To determine whether annexin 2 is required for the relocalization of temperature-sensitive v-Src, we used siRNA to deplete R1LA29 cells of annexin 2 prior to switching the cells to the permissive temperature. At the restrictive temperature, annexin 2-depleted cells were not morphologically different from wild-type cells, and there was no obvious difference in the staining intensity or localization of active Src, F-actin, or FAK-pY576 (Figs. 2A and 3). At the permissive temperature, however, cells lacking annexin 2 failed to exhibit the relocalization of active Src, FAK-pY576, paxillin-pY118, and actin to the perinuclear endosomal compartment, membrane ruffles, or macropinosomes, and the morphological changes associated with transformation did not occur. The cells remained large and flat with abundant stress fibers. Cells depleted of annexin 2 still contained active Src, as observed on Western blotting (Fig. 2B), but this was almost entirely localized at focal adhesions (see white arrowheads at the 24-h time point in Fig. 2A). Fig. 2 (C and D) shows histograms representing the proportion of Src phosphorylation on tyrosine 416 observed at various time points and the efficiency of annexin 2 knockdown achieved at each time point. There was no statistically significant difference in the kinetics of v-Src phosphorylation (believed to be an auto-catalytic event) between control and annexin 2-depleted cells, suggesting that v-Src activation alone is insufficient to elicit the morphological changes associated with transformation.


Annexin 2 has a dual role as regulator and effector of v-Src in cell transformation.

Hayes MJ, Moss SE - J. Biol. Chem. (2009)

Membrane targeting of activated Src requires annexin 2. A, R1LA29 cells were depleted of annexin 2 by siRNA treatment for 4 days at 41 °C. Cultures were then moved to 35 °C for the times indicated prior to fixation and immunostaining for annexin 2 (anx 2), Src-pY416, and F-actin. The top panel shows confocal sections at the base of cells grown at 41 °C, with both annexin 2-positive and annexin 2-negative cells in the field of view. Annexin 2 is present throughout the cytoplasm, the actin cytoskeleton is dominated by stress fibers, and Src-pY416 is undetectable. After 4 h at 35 °C, annexin 2, actin, and Src-pY416 appear at the actin-rich cortex and in a perinuclear vesicular domain (pink arrowheads), whereas there is no appearance of Src-pY416 at these sites and no change in cell shape in annexin 2-depleted cells. After 24 h, Src-pY416 is clearly enriched at the focal adhesions (white arrowheads) in annexin 2-depleted cells, but there is no morphological transformation. Insets show diagrammatic representations of the confocal plane. Scale bars, 25 μm. B, whole cell extracts were prepared from R1LA29 cells following a 4-day culture at 41 °C in the presence of control or annexin 2 siRNA and from cells that had been switched to the permissive temperature for the times indicated and immunoblotted as described in the legend to Fig. 1B. The right panel shows extracts from the control siRNA-treated cells at the restrictive temperature and after 36 h at the permissive temperature. There is an increase in phosphorylation of Src on phospho-Tyr416 and of paxillin on phospho-Tyr118, but phosphorylation on FAK at phospho-Tyr576 is impaired. C, densitometric analysis of Src-pY416 Western blots in control and annexin 2-depleted cells shows that the band intensity, indicative of Src activation, increases in a similar manner under both experimental conditions. KD, knockdown. D, a densitometric analysis shows that annexin 2 levels are maintained at least 50% below the control (Ctrl; when normalized to tubulin) for the duration of the experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2665074&req=5

fig2: Membrane targeting of activated Src requires annexin 2. A, R1LA29 cells were depleted of annexin 2 by siRNA treatment for 4 days at 41 °C. Cultures were then moved to 35 °C for the times indicated prior to fixation and immunostaining for annexin 2 (anx 2), Src-pY416, and F-actin. The top panel shows confocal sections at the base of cells grown at 41 °C, with both annexin 2-positive and annexin 2-negative cells in the field of view. Annexin 2 is present throughout the cytoplasm, the actin cytoskeleton is dominated by stress fibers, and Src-pY416 is undetectable. After 4 h at 35 °C, annexin 2, actin, and Src-pY416 appear at the actin-rich cortex and in a perinuclear vesicular domain (pink arrowheads), whereas there is no appearance of Src-pY416 at these sites and no change in cell shape in annexin 2-depleted cells. After 24 h, Src-pY416 is clearly enriched at the focal adhesions (white arrowheads) in annexin 2-depleted cells, but there is no morphological transformation. Insets show diagrammatic representations of the confocal plane. Scale bars, 25 μm. B, whole cell extracts were prepared from R1LA29 cells following a 4-day culture at 41 °C in the presence of control or annexin 2 siRNA and from cells that had been switched to the permissive temperature for the times indicated and immunoblotted as described in the legend to Fig. 1B. The right panel shows extracts from the control siRNA-treated cells at the restrictive temperature and after 36 h at the permissive temperature. There is an increase in phosphorylation of Src on phospho-Tyr416 and of paxillin on phospho-Tyr118, but phosphorylation on FAK at phospho-Tyr576 is impaired. C, densitometric analysis of Src-pY416 Western blots in control and annexin 2-depleted cells shows that the band intensity, indicative of Src activation, increases in a similar manner under both experimental conditions. KD, knockdown. D, a densitometric analysis shows that annexin 2 levels are maintained at least 50% below the control (Ctrl; when normalized to tubulin) for the duration of the experiment.
Mentions: Annexin 2 Is Required for Plasma Membrane Targeting of v-Src—To determine whether annexin 2 is required for the relocalization of temperature-sensitive v-Src, we used siRNA to deplete R1LA29 cells of annexin 2 prior to switching the cells to the permissive temperature. At the restrictive temperature, annexin 2-depleted cells were not morphologically different from wild-type cells, and there was no obvious difference in the staining intensity or localization of active Src, F-actin, or FAK-pY576 (Figs. 2A and 3). At the permissive temperature, however, cells lacking annexin 2 failed to exhibit the relocalization of active Src, FAK-pY576, paxillin-pY118, and actin to the perinuclear endosomal compartment, membrane ruffles, or macropinosomes, and the morphological changes associated with transformation did not occur. The cells remained large and flat with abundant stress fibers. Cells depleted of annexin 2 still contained active Src, as observed on Western blotting (Fig. 2B), but this was almost entirely localized at focal adhesions (see white arrowheads at the 24-h time point in Fig. 2A). Fig. 2 (C and D) shows histograms representing the proportion of Src phosphorylation on tyrosine 416 observed at various time points and the efficiency of annexin 2 knockdown achieved at each time point. There was no statistically significant difference in the kinetics of v-Src phosphorylation (believed to be an auto-catalytic event) between control and annexin 2-depleted cells, suggesting that v-Src activation alone is insufficient to elicit the morphological changes associated with transformation.

Bottom Line: Here, we report that this is dependent on annexin 2, a v-Src substrate and calcium-dependent regulator of actin dynamics.In cells depleted of annexin 2 by small interfering RNA, v-Src becomes activated at the permissive temperature but does not target to the plasma membrane or to perinuclear vesicles, and cell transformation does not occur.Our findings reveal a dual role for annexin 2, first as a regulator of v-Src trafficking and targeting and second as a v-Src effector in the reorganization of actin.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, University College London Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, United Kingdom.

ABSTRACT
Cell transformation by v-Src involves rearrangement of the actin cytoskeleton, disassembly of focal adhesions, and the development of anchorage-independent growth. Here, we report that this is dependent on annexin 2, a v-Src substrate and calcium-dependent regulator of actin dynamics. Using a thermoactivatable mutant of v-Src, we show that at the permissive temperature, annexin 2 becomes phosphorylated and colocalizes with activated v-Src and focal adhesion kinase both at the plasma membrane and in a Rab11-positive compartment of the endosomal pathway. In cells depleted of annexin 2 by small interfering RNA, v-Src becomes activated at the permissive temperature but does not target to the plasma membrane or to perinuclear vesicles, and cell transformation does not occur. Our findings reveal a dual role for annexin 2, first as a regulator of v-Src trafficking and targeting and second as a v-Src effector in the reorganization of actin.

Show MeSH
Related in: MedlinePlus