ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization
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eLife assessment
The manuscript describes a fundamental study of the atypical MAPK, ERK3, in the activation of RhoGTPase Cdc42 and the formation of actin-rich protrusions and cell migration. The results show that ERK3 is required for the motility of tumor cells in vivo, providing a new target for fighting metastasis.
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Abstract
The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding-proteins and nucleation-promoting factors to perform fundamental cellular functions. We have previously shown that ERK3, an atypical MAPK, controls IL-8 production and chemotaxis (Bogueka et al., 2020). Here, we show in human cells that ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent RAC1 and CDC42 activation, maintenance of F-actin content, filopodia formation, and epithelial cell migration. Further, ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. ERK3 kinase activity was required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.
Article activity feed
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The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique …
The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.
I really enjoyed reading this paper - what a great story! It really highlights how little we still know about actin regulation (even in human cells!), but it also does a great job filling in some of that knowledge with this pathway involving this atypical MAPK.
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S189 mutants (ERK3 S189A/ERK3 S189D)
Super interesting! Do these mutants have motility defects too?
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Interestingly, we readily detected the ARP2/3 complex subunits ARP3, ARP2 and ARPC1A as well as ERK3 by immunoblots in active Rac1/Cdc42 pull-downs
Very cool! Did you check other subunits or did they not precipitate?
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These data suggest that ERK3 could function as nucleation promoting factor to promote ARP2/3-dependent actin polymerization.
Woah! Curious if the kinase activity (that you test in the next section) is required for this change in actin polymerization?
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These results were corroborated by the colocalization of ERK3 with Cdc42 to the protrusions at the cell leading-edge
It would be interesting to see this in ERK3 knockdowns as well to see if the localization of Cdc42 changes with loss of ERK3. I'm also curious about the localization of Rac1.
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these data suggest that ERK3 likely controls actin cytoskeleton dynamics thereby influencing cell shape, motility and polarized migration.
By eye, I agree that it looks like cell shape is altered in Figure 1A, but it would be cool to include a quantification (maybe like how round the cells are or something). I also think that Figures 1H-J are relevant here to help connect some dots for this conclusion and show that the loss of ERK3 does cause changes in specific actin dynamics that result in shape and motility defects, but you don't talk about Fig 1H-J until later. Finally, I think the conclusion about polarized migration is more relevant in the next section than at the end of this section.
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ERK3 knockdown significantly decreased levels of both basal and EGF-induced GTP-bound Cdc42 and Rac1 in primary (HMEC)
Are there stats for Figure 2F? It seems like there's more variance with the Rac1 blot quantifications than with the Cdc42 blot quantifications.
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S418D-overexpressing cells exhibited F-actin-rich protrusions
It might be useful to quantify protrusions for all of 6F because it seems like there are definitely some big differences!
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These experiments showed that although in cultured cells ERK3 regulated the activity of both Cdc42 and Rac1, it only directly stimulated the GDP-GTP exchange of Cdc42
I find this interesting also in terms of the effects of ERK3 knockdown on the morphology of the cells and the specific actin structures. You mentioned previously that Rac1 leads to the formation of lamellipodia and Cdc42 leads to the formation of filopodia, and looking at the images of your cells, it looks like filopodia are more affected than lamellipodia. Is that something you noticed looking through your images?
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Actin is one of the most abundant and highly conserved proteins with over 95% homology among all isoforms
You might specify that this is true for human isoforms. There are lots of weird actins out there in other organisms that are well under that 95%
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eLife assessment
The manuscript describes a fundamental study of the atypical MAPK, ERK3, in the activation of RhoGTPase Cdc42 and the formation of actin-rich protrusions and cell migration. The results show that ERK3 is required for the motility of tumor cells in vivo, providing a new target for fighting metastasis.
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Reviewer #1 (Public Review):
Previously the authors showed that ERK3 plays a critical role in the production of IL-8, immune cell chemotaxis, and metastasis (Bogucka et al, eLife 2020). This is a follow-up study on these observations in which they uncover a critical role for ERK3 in the activation of RhoGTPases, formation of actin-rich protrusions, and actin polymerization. Previous publications have reported a critical role of ERK3 in regulating cell morphology and migration. However, the molecular mechanisms responsible for these phenotypes remain elusive. The polarized phenotype of motile cells involves complex actin cytoskeleton re-arrangements, and in this study, the authors demonstrate a direct role for MAPK6 kinase in regulating actin dynamics.
First, the authors confirm that loss of ERK3 negatively affects MDA-MB231 cell …
Reviewer #1 (Public Review):
Previously the authors showed that ERK3 plays a critical role in the production of IL-8, immune cell chemotaxis, and metastasis (Bogucka et al, eLife 2020). This is a follow-up study on these observations in which they uncover a critical role for ERK3 in the activation of RhoGTPases, formation of actin-rich protrusions, and actin polymerization. Previous publications have reported a critical role of ERK3 in regulating cell morphology and migration. However, the molecular mechanisms responsible for these phenotypes remain elusive. The polarized phenotype of motile cells involves complex actin cytoskeleton re-arrangements, and in this study, the authors demonstrate a direct role for MAPK6 kinase in regulating actin dynamics.
First, the authors confirm that loss of ERK3 negatively affects MDA-MB231 cell motility and migration, both in vitro and in vivo. Interestingly loss of ERK3 reduced F-actin content in primary breast mammary epithelial cells. The authors used a multi-disciplinary approach to elucidate the underlying mechanisms. Using biochemical methods, they elegantly show the direct link between ERK3 and RhoGTPases as well as the ARP2/3 complex. Furthermore, direct binding of ERK3 to Rac1, Cdc42, and Arp2/3 complex is shown by biochemical assays, and these observations are validated by monitoring the interaction between ERK3 and the Cdc42/ARP2/3 complex in cells at endogenous levels. The finding that ERK3 acts as a GEF for Cdc42 and not Rac1 is interesting and further links this kinase to PAKs. PAK kinases have been shown to phosphorylate ERK3 at Ser 189 in the SEG motif to activate ERK3 (Deleris et al JBC,2011). Overall, this study generated a lot of interesting data and the work has been well-executed and properly interpreted. The main findings are novel and important, and they are of particular interest to readers in the fields of cell migration and actin dynamics. This manuscript is also likely to stimulate additional investigations using biophysical and structural methods to further decipher GEF activity controls ERK3.
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**Reviewer #2 (Public Review):
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MAPKs are key fundamental enzymes and out of the 14 MAPKs, ERK3 and ERK4 remain less studied. The authors have made some interesting discoveries on ERK3, especially in the context of chemotaxis and tumourigenesis previously (Bogucka et al eLife 2020). Here they investigated the role of ERK3 in the control of cell architecture. Loss of ERK3 led to a reduction in the formation of actin-rich protrusions which led the authors logically to look for the activation of RhoGTPases. Intriguingly, they found that ERK3 functioned as a GEF for Cdc42 but not for Rac1. Further, they identified that Rac-WAVE and Arp2/3 were present at endogenous levels in a heteromeric complex in cells. As ERK3-deficient breast epithelial cells exhibit less F-actin content, this has led the authors to check for Arp2/3-dependent events …**Reviewer #2 (Public Review):
**
MAPKs are key fundamental enzymes and out of the 14 MAPKs, ERK3 and ERK4 remain less studied. The authors have made some interesting discoveries on ERK3, especially in the context of chemotaxis and tumourigenesis previously (Bogucka et al eLife 2020). Here they investigated the role of ERK3 in the control of cell architecture. Loss of ERK3 led to a reduction in the formation of actin-rich protrusions which led the authors logically to look for the activation of RhoGTPases. Intriguingly, they found that ERK3 functioned as a GEF for Cdc42 but not for Rac1. Further, they identified that Rac-WAVE and Arp2/3 were present at endogenous levels in a heteromeric complex in cells. As ERK3-deficient breast epithelial cells exhibit less F-actin content, this has led the authors to check for Arp2/3-dependent events here. By employing a variety of knockdown and complementation approaches, the authors convincingly demonstrate that the kinase activity of ERK3 is not required for the total F-actin content but for the formation of actin-rich protrusions. Finally, loss of ERK3 reduced random cell motility in vitro and in vivo, which was accomplished by intravital imaging of breast cancer cells in mice. Many protein kinases have catalysis-dependent and -independent functions (catalytic activity versus allosteric activity) and here is another example that deserves further investigation and opens new lines of investigation. -
Reviewer #3 (Public Review):
Bogucka-Janczi et al. have carefully dissected a role for ERK3 in the regulation of actin cytoskeleton dynamics. They identify two "nodes" of operation for ERK3 in this process, firstly, the interaction and effect of ERK3 on the small GTPases Rac1 and Cdc42, and secondly, the interaction with and effect of ERK3 on ARP3. In addition, they show a robust phosphorylation of ERK3-S189 in response to EGF stimulation. They further show that ERK3 knockdown results in a decrease of chemotaxis in response to EGF, although they have been unable to identify an important role of S189 phosphorylation in this context.
The authors have clearly carried out a large number of experiments in order to understand these complex events in a highly dynamic process. They have largely succeeded, although some aspects are rather unclear.
Reviewer #3 (Public Review):
Bogucka-Janczi et al. have carefully dissected a role for ERK3 in the regulation of actin cytoskeleton dynamics. They identify two "nodes" of operation for ERK3 in this process, firstly, the interaction and effect of ERK3 on the small GTPases Rac1 and Cdc42, and secondly, the interaction with and effect of ERK3 on ARP3. In addition, they show a robust phosphorylation of ERK3-S189 in response to EGF stimulation. They further show that ERK3 knockdown results in a decrease of chemotaxis in response to EGF, although they have been unable to identify an important role of S189 phosphorylation in this context.
The authors have clearly carried out a large number of experiments in order to understand these complex events in a highly dynamic process. They have largely succeeded, although some aspects are rather unclear.
-
S418D-overexpressing cells exhibited F-actin-rich protrusions
It might be useful to quantify protrusions for all of 6F because it seems like there are definitely some big differences!
-
These data suggest that ERK3 could function as nucleation promoting factor to promote ARP2/3-dependent actin polymerization.
Woah! Curious if the kinase activity (that you test in the next section) is required for this change in actin polymerization?
-
Interestingly, we readily detected the ARP2/3 complex subunits ARP3, ARP2 and ARPC1A as well as ERK3 by immunoblots in active Rac1/Cdc42 pull-downs
Very cool! Did you check other subunits or did they not precipitate?
-
These results were corroborated by the colocalization of ERK3 with Cdc42 to the protrusions at the cell leading-edge
It would be interesting to see this in ERK3 knockdowns as well to see if the localization of Cdc42 changes with loss of ERK3. I'm also curious about the localization of Rac1.
-
These experiments showed that although in cultured cells ERK3 regulated the activity of both Cdc42 and Rac1, it only directly stimulated the GDP-GTP exchange of Cdc42
I find this interesting also in terms of the effects of ERK3 knockdown on the morphology of the cells and the specific actin structures. You mentioned previously that Rac1 leads to the formation of lamellipodia and Cdc42 leads to the formation of filopodia, and looking at the images of your cells, it looks like filopodia are more affected than lamellipodia. Is that something you noticed looking through your images?
-
ERK3 knockdown significantly decreased levels of both basal and EGF-induced GTP-bound Cdc42 and Rac1 in primary (HMEC)
Are there stats for Figure 2F? It seems like there's more variance with the Rac1 blot quantifications than with the Cdc42 blot quantifications.
-
S189 mutants (ERK3 S189A/ERK3 S189D)
Super interesting! Do these mutants have motility defects too?
-
these data suggest that ERK3 likely controls actin cytoskeleton dynamics thereby influencing cell shape, motility and polarized migration.
By eye, I agree that it looks like cell shape is altered in Figure 1A, but it would be cool to include a quantification (maybe like how round the cells are or something). I also think that Figures 1H-J are relevant here to help connect some dots for this conclusion and show that the loss of ERK3 does cause changes in specific actin dynamics that result in shape and motility defects, but you don't talk about Fig 1H-J until later. Finally, I think the conclusion about polarized migration is more relevant in the next section than at the end of this section.
-
Actin is one of the most abundant and highly conserved proteins with over 95% homology among all isoforms
You might specify that this is true for human isoforms. There are lots of weird actins out there in other organisms that are well under that 95%
-
The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique …
The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.
I really enjoyed reading this paper - what a great story! It really highlights how little we still know about actin regulation (even in human cells!), but it also does a great job filling in some of that knowledge with this pathway involving this atypical MAPK.
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