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Dorsal Ruffles Enhance Activation of Akt by Growth Factors

Sei Yoshida, Regina Pacitto, Catherine Sesi, Leszek Kotula, View ORCID ProfileJoel A. Swanson
doi: https://doi.org/10.1101/324434
Sei Yoshida
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620
2Center for Live-Cell Imaging (CLCI), University of Michigan Medical School, Ann Arbor, MI 48109-5620
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  • For correspondence: seiyoshi@umich.edu jswan@umich.edu
Regina Pacitto
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620
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Catherine Sesi
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620
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Leszek Kotula
3Department of Urology, SUNY Upstate Medical School, Syracuse, NY 13210
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Joel A. Swanson
1Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109-5620
2Center for Live-Cell Imaging (CLCI), University of Michigan Medical School, Ann Arbor, MI 48109-5620
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  • ORCID record for Joel A. Swanson
  • For correspondence: seiyoshi@umich.edu jswan@umich.edu
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Summary

In fibroblasts, platelet-derived growth factor (PDGF) stimulates macropinocytosis and PI 3-kinase (PI3K)-dependent phosphorylation of Akt, leading to activation of mTORC1, a protein complex controlling metabolism and cell growth. PIP3, the phosphoinositide product of PI3K that activates Akt, is frequently concentrated within the macropinocytic cups of growth factor-stimulated cells, which suggests that cup structure enhances phosphorylation of Akt by facilitating PI3K activity. However, inhibitors of the cytoskeleton which block cup formation do not reduce Akt phosphorylation in response to high concentrations of PDGF. Because the dynamics of Akt phosphorylation after stimulation by PDGF can differ from those that follow stimulation with epidermal growth factor (EGF), we analyzed the contributions of the actin and microtubule cytoskeleton to activation of Akt by these two growth factors. Actin-rich, circular dorsal ruffles (CDR), analogous to macropinocytic cups, appeared within several minutes of adding EGF or PDGF and often closed to form macropinosomes. Nocodazole, an inhibitor of microtubule polymerization, blocked both PDGF- and EGF-induced CDR formation, and inhibited phosphorylation of Akt in response to EGF but not PDGF. At concentrations that saturate their cognate receptors, EGF stimulated lower maximal levels of Akt phosphorylation than did PDGF. We hypothesized that weak signals elicited by EGF receptors require cytoskeleton-dependent amplification of PI3K for maximal phosphorylation of Akt. In both PDGF- and EGF-stimulated cells, quantitative immunofluorescence showed increased Akt phosphorylation in cells containing CDR, with PIP3 and Akt concentrated in CDR and ruffles. Stimulation with low concentrations of PDGF elicited lower levels of Akt phosphorylation, which, like responses to EGF, were inhibited by nocodazole. These results indicate that when receptor signaling generates low levels of PI3K activity, CDR facilitate local amplification of PI3K, PIP3 synthesis and phosphorylation of Akt.

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Posted May 16, 2018.
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Dorsal Ruffles Enhance Activation of Akt by Growth Factors
Sei Yoshida, Regina Pacitto, Catherine Sesi, Leszek Kotula, Joel A. Swanson
bioRxiv 324434; doi: https://doi.org/10.1101/324434
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Dorsal Ruffles Enhance Activation of Akt by Growth Factors
Sei Yoshida, Regina Pacitto, Catherine Sesi, Leszek Kotula, Joel A. Swanson
bioRxiv 324434; doi: https://doi.org/10.1101/324434

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