JNK-mediated Phosphorylation of Paxillin in Adhesion Assembly and Tension-induced Cell Death by the Adenovirus Death Factor E4orf4

Josée N Lavoie,Claudia Champagne,Nicolas Smadja-Lamére,Philip E Branton,Marie-Chloé Boulanger

doi:10.1074/jbc.m803364200

Josée N Lavoie, Claudia Champagne + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m803364200

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Abstract

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.

Highlights

We provide strong evidence that dysregulation of adhesion dynamics downstream of a SrcRho-ROCK-Jun N-terminal kinases (JNKs) pathway is an important component of the death signaling pathway, which appears to involve a chronic increase in internal force generation
The chronic increase in cytoskeletal tension generated by the co-assembly of robust adhesions and tensile actin fibers would cause the breakage of adhesions and the sudden collapse of the contractile perinuclear actin network, which precedes the onset of cell blebbing in early region 4 ORF4 (E4orf4)-expressing cells [14]
It is reasonable to think that one of the major lesions induced by E4orf4 in tumor cells is the generation of a high level of internal cell tension combined with deregulation of the cell traction forces, which would be driven by the concerted action of myosin II and JNK downstream of ROCK

Summary

The abbreviations used are

E4orf, early region 4 ORF4; Ad2 E4orf, adenovirus type 2 early region 4 open reading frame 4; AP-1, activator protein 1; EV, empty vector; FA, focal adhesion; FAK, FA kinase; FRAP, fluorescence recovery after photobleaching; GAP, GTPase-activating protein; GFP, green fluorescent protein; MAP, mitogen-activated protein; siRNA, small interfering RNA; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; JNK, c-Jun N-terminal kinase; mRFP, monomeric red fluorescent protein; ROCK, Rho kinase; N-Wasp; Tam, dominant-negative transactivation deletion mutant of c-Jun; WT, wild-type; ERK, extracellular signal-regulated kinase; ROI, region of interest; Mf, mobile fraction; p-, phospho-. Because inhibition of myosin II can provide a marked protection against E4orf killing, we have proposed that some orderly lesions caused by increased cytoskeletal tension might contribute to engage the cell death pathway given that myosin II is the major motor protein responsible for the generation of internal cell tension [14, 15] In this scenario, assembly of the peculiar juxtanuclear contractile network would be a consequence of the initial and lethal changes in actin dynamics rather than a cause of cell death. This is the case for focal adhesion kinase (FAK) and ERK, two pathways providing critical survival signals in tumor cells [13, 17, 18] Based on these findings, we have proposed that the resulting unbalance in Src signals might trigger the death of transformed and tumor cells expressing E4orf. We propose a model whereby E4orf perturbs tensional homeostasis in tumor cells by hijacking a Src-Rho-ROCK pathway that controls cell tension and adhesion dynamics via the concerted action of myosin II and JNK/paxillin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION