Cell Density-Dependent Increase in Tyrosine-Monophosphorylated ERK2 in MDCK Cells Expressing Active Ras or Raf

Abstract

The extracellular signal-regulated kinase (ERK) is one of the principal hub proteins that transmit growth signals from upstream oncogene products including Ras and BRaf to downstream effector proteins. However, there are both reports supporting and refuting the increase in ERK activity in cancer tissues expressing the active Ras and BRaf proteins. We considered that the cell density might account for this discrepancy. To examine this possibility, we prepared Madin-Darby canine kidney (MDCK) cells that expressed an active HRas, NRas, KRas, or BRaf and an ERK biosensor based on the principle of Förster resonance energy transfer (FRET). As we anticipated, expression of the active Ras or BRaf increased ERK activity at low cell densities. However, the ERK activity was markedly suppressed at high cell densities irrespective of the expression of the active Ras or BRaf. Western blotting analysis with Phos-tag gel revealed the decrease of tyrosine and threonine-diphosphorylated active ERK and the increase of tyrosine-monophosphorylated inactive ERK at high cell density. In addition, we found that calyculin A, an inhibitor for PPP-subfamily protein serine/threonine phosphatases, decreased the tyrosine-monophosphorylated ERK. Our study suggests that PPP-subfamily phosphatases may be responsible for cell density-dependent ERK dephosphorylation in cancer cells expressing active Ras or BRaf protein.

Dying under pressure: cellular characterisation and in vivo functions of cell death induced by compaction

“…The sensing of deformations is triggered through the modulation of ERK activity which will impact cell survival via the inhibition of the pro-apoptotic protein Hid [Moreno et al, 2018]. Those results are in agreement with the previous reports of ERK activation by cellular tension in cell culture [Tschumperlin et al, 2004, Aoki et al, 2013, Hirata et al, 2015, Kawabata and Matsuda, 2016, Aoki et al, 2017 and in vivo [Sano et al, 2016]. Finally, even if they have not been formally associated with compaction-driven death yet, other pathways which regulate cell survival and can be modulated by mechanical forces may also be relevant for compaction-driven cell death.…”

Effect of pharmacological modulation of actin and myosin on collective cell electrotaxis

“…In contrast to the effect of lowering the contractility, increasing the level of contractility by treating the monolayers with 5 nM calyculin A [Kawabata and Matsuda, ] significantly attenuated cathode‐directed electrotaxis of the MDCK monolayers ( d = 0.12, interaction effect P < 0.01, 2‐way ANOVA). Calyculin A inhibits protein phosphatases and thus promotes myosin activity by inhibiting myosin light chain phosphatase [Goeckeler and Wysolmerski, ; Stull et al, ].…”

Mechanisms of inside-out signaling of the high-affinity IgG receptor FcγRI

“…Subsequently, beads were washed in Triton lysis buffer and boiled in reducing sample buffer. Samples were loaded onto a 12% polyacrylamide separating gel (no Phos-tag) and a 7.5% polyacrylamide separating gel containing 100 M Phos-tag (Wako Chemicals) and 200 M MnCl 2 (50,51). After electrophoresis, the Phos-tag gel was washed in a 1 mM EDTA solution, followed by a wash with EDTA-free solution, before transferring the proteins to a polyvinylidene difluoride membrane.…”

“…Antibody-dependent cellular cytotoxicity ADCC of 51 Cr-labeled target cells was described previously (52). Briefly, 1 × 10 6 target cells were labeled with 100 Ci (3.7 MBq) 51 Cr for 2 hours. After extensive washing, cells were adjusted to 10 5 /ml.…”