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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.

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Relocalization of FAK-pY576 in response to v-Src-mediated cell transformation is dependent upon annexin 2. R1LA29 cells were partially depleted of annexin 2 as described in the legend to Fig. 5, fixed, and immunostained for annexin 2 (anx 2), FAK-pY576, and F-actin in both the untransformed and transformed states. Following the switch from restrictive to permissive temperature for 1 h, annexin 2-depleted cells (pink arrowheads) remained large and flat with numerous focal adhesions enriched in FAK-pY576. In contrast, annexin 2-positive cells acquired a transformed phenotype in which annexin 2 relocalized to the cell cortex. By 8 h, there was a dramatic increase in the cellular content of FAK-pY576 in annexin 2-positive cells, in which it relocalized to the annexin 2-rich cortex and to the perinuclear annexin 2-rich and actin-rich vesicles (white arrowheads). Relocalization of FAK-pY576 did not occur in annexin 2-depleted cells. Scale bars, 20 μm.
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fig3: Relocalization of FAK-pY576 in response to v-Src-mediated cell transformation is dependent upon annexin 2. R1LA29 cells were partially depleted of annexin 2 as described in the legend to Fig. 5, fixed, and immunostained for annexin 2 (anx 2), FAK-pY576, and F-actin in both the untransformed and transformed states. Following the switch from restrictive to permissive temperature for 1 h, annexin 2-depleted cells (pink arrowheads) remained large and flat with numerous focal adhesions enriched in FAK-pY576. In contrast, annexin 2-positive cells acquired a transformed phenotype in which annexin 2 relocalized to the cell cortex. By 8 h, there was a dramatic increase in the cellular content of FAK-pY576 in annexin 2-positive cells, in which it relocalized to the annexin 2-rich cortex and to the perinuclear annexin 2-rich and actin-rich vesicles (white arrowheads). Relocalization of FAK-pY576 did not occur in annexin 2-depleted cells. Scale bars, 20 μm.

Mentions: Redistribution of Annexin 2, v-Src, and FAK-pY576 during Transformation—To test the hypothesis that a functional relationship may exist between annexin 2, Src, and FAK, we used the well characterized Rat-1 embryonic fibroblast model of v-Src transformation that expresses the thermoactivatable LA29 mutant of Src (R1LA29 cells). In agreement with other studies, we found that when cells were cultured at 41 °C (the nonpermissive temperature), the F-actin cytoskeleton appeared to be predominantly organized into basal stress fibers, with the cells flattened and apparently contact-inhibited (27). Annexin 2 is distributed throughout the cytoplasm in such cells but is excluded from the nucleus, and there is little detectable staining of Src-pY416 (Fig. 1A). Phosphorylation of Src at tyrosine 416 (Src-pY416) stabilizes the protein in an open conformation (25) and is indicative of its activity. Over a 48-h time course following a switch to the permissive temperature (35 °C), annexin 2 became enriched at the cell cortex, and Src-pY416 was observed at the focal adhesions (not visible in Fig. 1, which shows a confocal section through the middle of the cell, but clear in Fig. 3A) and then also the plasma membrane. Transformation was also accompanied by dramatic rearrangement of the F-actin cytoskeleton and a change in cell shape. Thus, cells switched from a flattened, stress fiber-dominated phenotype to become rounder, elongated, or fusiform with cortical F-actin. Western blot analysis revealed rapid appearance of Src-pY416, FAK-pY925, and paxillin-pY118 at 1 h (Fig. 1B). Phosphorylation of FAK on tyrosine 576 (in the kinase domain activation loop) appeared somewhat later. In some experiments, we observed an apparent biphasic peak of phosphorylation of Src and FAK-pY925 (as shown in these blots), but this was not seen consistently. We did not observe tyrosine phosphorylation of annexin 2 in cells at 41 °C, but after 4 h at 35 °C, it became detectable and was strongly phosphorylated by 18 h (Fig. 1C). These findings are consistent with published reports of annexin 2 tyrosine phosphorylation by the temperature-sensitive NY68 Src mutant (35, 36). Interestingly, the relocalization of annexin 2 to the cortex is evident even after 1 h at the permissive temperature (see example in Fig. 3), suggesting that annexin 2 translocates before it becomes tyrosine-phosphorylated. By 48 h, many of the cells showed marked enrichment of Src-pY416 and annexin 2 in the cortex, in ruffles, on large macropinosomes, and on perinuclear vesicles (Fig. 1A, inset). When we examined R1LA29 cells transiently transfected with a GFP fusion of the temperature-sensitive LA29 Src mutant, we observed migration of v-Src-GFP from what appeared to be focal adhesions (short, linear, and basal structures at the periphery of the cell) to large macropinosomes when they were grown at the permissive temperature (supplemental Fig. 1 and movie). The protein appeared to spool off the focal adhesion onto the nascent macropinosome. Similar results were observed for c-Src-GFP (data not shown).


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

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

Relocalization of FAK-pY576 in response to v-Src-mediated cell transformation is dependent upon annexin 2. R1LA29 cells were partially depleted of annexin 2 as described in the legend to Fig. 5, fixed, and immunostained for annexin 2 (anx 2), FAK-pY576, and F-actin in both the untransformed and transformed states. Following the switch from restrictive to permissive temperature for 1 h, annexin 2-depleted cells (pink arrowheads) remained large and flat with numerous focal adhesions enriched in FAK-pY576. In contrast, annexin 2-positive cells acquired a transformed phenotype in which annexin 2 relocalized to the cell cortex. By 8 h, there was a dramatic increase in the cellular content of FAK-pY576 in annexin 2-positive cells, in which it relocalized to the annexin 2-rich cortex and to the perinuclear annexin 2-rich and actin-rich vesicles (white arrowheads). Relocalization of FAK-pY576 did not occur in annexin 2-depleted cells. Scale bars, 20 μm.
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Related In: Results  -  Collection

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fig3: Relocalization of FAK-pY576 in response to v-Src-mediated cell transformation is dependent upon annexin 2. R1LA29 cells were partially depleted of annexin 2 as described in the legend to Fig. 5, fixed, and immunostained for annexin 2 (anx 2), FAK-pY576, and F-actin in both the untransformed and transformed states. Following the switch from restrictive to permissive temperature for 1 h, annexin 2-depleted cells (pink arrowheads) remained large and flat with numerous focal adhesions enriched in FAK-pY576. In contrast, annexin 2-positive cells acquired a transformed phenotype in which annexin 2 relocalized to the cell cortex. By 8 h, there was a dramatic increase in the cellular content of FAK-pY576 in annexin 2-positive cells, in which it relocalized to the annexin 2-rich cortex and to the perinuclear annexin 2-rich and actin-rich vesicles (white arrowheads). Relocalization of FAK-pY576 did not occur in annexin 2-depleted cells. Scale bars, 20 μm.
Mentions: Redistribution of Annexin 2, v-Src, and FAK-pY576 during Transformation—To test the hypothesis that a functional relationship may exist between annexin 2, Src, and FAK, we used the well characterized Rat-1 embryonic fibroblast model of v-Src transformation that expresses the thermoactivatable LA29 mutant of Src (R1LA29 cells). In agreement with other studies, we found that when cells were cultured at 41 °C (the nonpermissive temperature), the F-actin cytoskeleton appeared to be predominantly organized into basal stress fibers, with the cells flattened and apparently contact-inhibited (27). Annexin 2 is distributed throughout the cytoplasm in such cells but is excluded from the nucleus, and there is little detectable staining of Src-pY416 (Fig. 1A). Phosphorylation of Src at tyrosine 416 (Src-pY416) stabilizes the protein in an open conformation (25) and is indicative of its activity. Over a 48-h time course following a switch to the permissive temperature (35 °C), annexin 2 became enriched at the cell cortex, and Src-pY416 was observed at the focal adhesions (not visible in Fig. 1, which shows a confocal section through the middle of the cell, but clear in Fig. 3A) and then also the plasma membrane. Transformation was also accompanied by dramatic rearrangement of the F-actin cytoskeleton and a change in cell shape. Thus, cells switched from a flattened, stress fiber-dominated phenotype to become rounder, elongated, or fusiform with cortical F-actin. Western blot analysis revealed rapid appearance of Src-pY416, FAK-pY925, and paxillin-pY118 at 1 h (Fig. 1B). Phosphorylation of FAK on tyrosine 576 (in the kinase domain activation loop) appeared somewhat later. In some experiments, we observed an apparent biphasic peak of phosphorylation of Src and FAK-pY925 (as shown in these blots), but this was not seen consistently. We did not observe tyrosine phosphorylation of annexin 2 in cells at 41 °C, but after 4 h at 35 °C, it became detectable and was strongly phosphorylated by 18 h (Fig. 1C). These findings are consistent with published reports of annexin 2 tyrosine phosphorylation by the temperature-sensitive NY68 Src mutant (35, 36). Interestingly, the relocalization of annexin 2 to the cortex is evident even after 1 h at the permissive temperature (see example in Fig. 3), suggesting that annexin 2 translocates before it becomes tyrosine-phosphorylated. By 48 h, many of the cells showed marked enrichment of Src-pY416 and annexin 2 in the cortex, in ruffles, on large macropinosomes, and on perinuclear vesicles (Fig. 1A, inset). When we examined R1LA29 cells transiently transfected with a GFP fusion of the temperature-sensitive LA29 Src mutant, we observed migration of v-Src-GFP from what appeared to be focal adhesions (short, linear, and basal structures at the periphery of the cell) to large macropinosomes when they were grown at the permissive temperature (supplemental Fig. 1 and movie). The protein appeared to spool off the focal adhesion onto the nascent macropinosome. Similar results were observed for c-Src-GFP (data not shown).

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