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.

The EGFR-Ras-ERK pathway is a major growth signaling cascade in many human cancers. Consequently, constitutive activation of Ras, which is found in 30% of human cancers, is believed to lead to constitutive ERK activation and, thereby, uncontrolled cell growth. Recently, we have reported that the cell growth is controlled not only by the basal ERK activity but also by the frequency of stochastic ERK activity pulses. In the present study, we have evaluated the effect of Ras on the temporal dynamics of Erk activity by using fluorescent resonance energy transfer (FRET) biosensors. We found that, in Madin-Darby Canine kidney (MDCK) cells expressing constitutively active HRAS (HRASV12), ERK activity stimulated by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), was markedly attenuated in comparison with the parent MDCK cells. Furthermore, the stochastic ERK activity pulses observed in the parent MDCK cells were almost completely abolished in the HRASV12-expressing MDCK cells. These observations have revealed that the temporal regulation of ERK activity is significantly different between normal and HRASV12-expressing cells. Citation Format: Noriyuki Kawabata, Michiyuki Matsuda. Constitutively-active HRAS suppresses stochastic Erk activity pulses in Madin-Darby canine kidney cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A13. doi: 10.1158/1557-3125.RASONC14-A13