Unravelling the Mechanism of TrkA-Induced Cell Death by Macropinocytosis in Medulloblastoma Daoy Cells

Abstract

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser¹⁸⁵ by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00255-16.

ACKNOWLEDGMENTS

We thank the many investigators who provided the following constructs for the experiments described here: M. Park (McGill University, Montreal, Canada) for GST-GGA3; A. Richmond (Vanderbilt University, TN) for GST-RhoRBD and DN and CA RhoB; A. Hall (Sloane-Kettering, NY) for GST-PAK-CRIB; D. Shalloway (University of California, Berkeley, CA) for GST-Ras RBD; T. Balla (NIH, MD) for PH domains of AKT and PLCδ as well as the RBD-CRD domains of c-Raf-1 fused with EGFP; Y. Sako (RIKEN ASI, Japan) for the CRD c-Raf-1 R⁸⁹A mutant; J. Donaldson (NIH, MD) for DN and CA-Arf6; P. Stork (Vollum Institute, OR) for DN and CA-Rap1b; N. Yamaguchi (Chiba University, Japan) for WT and DN Src; S. Ferguson (University of Ottawa, Ontario, Canada) for CA (G¹²V) and DN (T¹⁷N) Cdc42, DN (S¹⁷N) Rac, DN (K⁴⁴A) dynamin 2, DN (T³¹N) Arf1, DN (N¹⁹L) RhoA, DN (S³⁴N) Rab5, DN (T²²N) Rab7, and EGFP-tagged clathrin; Alberto Luini (Institute of Protein Biochemistry, Naples, Italy) for DN (S¹⁴⁷A) CtBP1/BARS; T. Endo (Chiba University, Chiba, Japan) for DN (T⁶⁶N) and CA (Q¹¹¹L) Rab34; and A. Pradines (INSERM, France) for the RhoB (S¹⁸⁵A) mutant.

This research was supported with funds from an operating grant from the Cancer Research Society to S.O.M. and from the Canadian Institutes of Health Research to S.W.M. (MOP-GMX-93643) as well as from private donations.

S.O.M. conceived and supervised the project. C.L. performed confocal microscopy, cell culture, and biochemical experiments and data analyses. J.L., A.T., and J.I.S.M. performed cell culture and biochemical experiments and data analyses. C.S. performed confocal studies. S.H.P. and S.W.M. provided essential reagents and intellectual support.

We declare that we have no conflicts of interest.