ZAK activation at the collided ribosome

Article ZAK activation at the collided ribosome https://doi.org/10.1038/s41586-025-09772-8 Received: 1 July 2025 Accepted: 17 October 2025 Published online: xx xx xxxx Open access Check for updates Vienna L. Huso1,2,5, Shuangshuang Niu3,5, Marco A. Catipovic1,2, James A. Saba1,2, Timo Denk3, Eugene Park1,2, Jingdong Cheng4, Otto Berninghausen3, Thomas Becker3, Rachel Green1,2 ✉ & Roland Beckmann3 ✉ Ribosome collisions activate the ribotoxic stress response mediated by the MAP3K ZAK, which in turn regulates cell-fate consequences through downstream phosphorylation of the MAPKs p38 and JNK1. Despite the critical role of ZAK during cellular stress, a mechanistic and structural understanding of ZAK–ribosome interactions and how these lead to activation remain elusive. Here we combine biochemistry and cryo-electron microscopy to discover distinct ZAK–ribosome interactions required for constitutive recruitment and for activation. We find that upon induction of ribosome collisions, interactions between ZAK and the ribosomal protein RACK1 enable its activation by dimerization of its SAM domains at the collision interface. Furthermore, we discover how this process is negatively regulated by the ribosome-binding protein SERBP1 to prevent constitutive ZAK activation. Characterization of novel SAM variants as well as a known pathogenic variant of the SAM domain of ZAK supports a key role of the SAM domain in regulating kinase activity on and off the ribosome, with some mutants bypassing the ribosome requirement for ZAK activation. Collectively, our data provide a mechanistic blueprint of the kinase activity of ZAK at the collided ribosome interface. The ribosome translates mRNA into protein, often with multiple ribosomes on a given mRNA called polysomes. Ribosomes are also essential sensors of cellular stress and can alert the cell of nutrient deprivation2, damage to mRNAs and chemical insults that directly target and damage ribosomes3–5. Such cellular stresses cause ribosomes to stall on problematic mRNA, resulting in the lagging ribosome colliding with the stalled ribosome. These ribosome collisions are a key signal to activate both quality control pathways and broad stress signalling responses4,6. For one of these signalling pathways, the ribotoxic stress response (RSR), the mitogen-activated protein kinase kinase kinase (MAP3K) ZAKα (referred to hereafter as ZAK) has a central role in orchestrating the RSR7–9. Previous studies have demonstrated that ZAK interacts constitutively with ribosomes during unstressed conditions but becomes activated (via autophosphorylation) and is released from the ribosome upon cellular stresses that impair translation1,10. Although previous studies have argued that ribosome collisions are key determinants of ZAK activation1, other studies have suggested that both colliding and individually stalled ribosomes may be potent triggers10,11. In either case, ZAK activates the stress-activated protein kinases (SAPKs) p38 and JNK, leading to cell cycle arrest and/or apoptosis12–14. Kinase regulation often requires scaffold proteins15, and ZAK has been shown to interact with 14-3-3 proteins off the ribosome in a canonical phosphorylation-dependent manner downstream of activation10,12. Another scaffold is the receptor for activated C-kinase (RACK1), a conserved eukaryotic ribosomal protein on the head of the 40S subunit, which was first characterized for PKC activation and later argued to be a signalling hub for kinases including JNK16–19. Of note, RACK1 resides exactly at the collision interface and has a critical role in collision-mediated quality control events20–24, hinting that RACK1 could scaffold ZAK on the ribosome. Although much is known about ZAK signalling and its downstream consequences for cell fate12,13, the molecular determinants of the interaction of ZAK with the ribosome and an understanding of how these interactions mediate activation have remained enigmatic. Here we elucidate how ZAK is recruited to ribosomes, both in its basal state and in induced stress conditions, and show how collision-specific interactions organized on RACK1 mediate ZAK activation. ZAK enrichment on ribosomes A single mammalian cell contains approximately 5 million ribosomes, and the ratio of ZAK to ribosomes is estimated at approximately 1:100 based on copy numbers measured across various cell types25,26. Therefore, we turned to overexpression of N-terminally tagged ZAK in HEK293T cells to enrich ZAK-bound ribosomes. We characterized ribosome binding of wild-type (WT) ZAK and monitored its phosphorylation status by Phos-tag immunoblotting after sucrose gradient fractionation. Overexpressed WT ZAK was found in the top fractions of the gradient and migrated on western blot (Phos-tag) at a molecular weight of approximately 250 kDa, consistent with fully phosphorylated (P) ‘activated’ protein1,12 (Fig. 1a); in addition, JNK-P levels were increased under basal conditions (Extended Data Fig. 1a). These observations reflect activation of ZAK and the RSR upon ZAK overexpression. We next overexpressed a kinase inactive ZAK with mutations in the activation loop: T161A/S165A27. Despite its strong overexpression, this Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 2Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA. 3Department of Biochemistry, Gene Center, University of Munich, Munich, Germany. 4Minhang Hospital & Institutes of Biomedical Sciences, Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism, Fudan University, Shanghai, China. 5These authors contributed equally: Vienna L. Huso, Shuangshuang Niu. ✉e-mail: ; 1 Nature | www.nature.com | 1 Article a A260 1.5 Free 80S b Polysomes 1 E/E 0.5 Fraction: WT ZAK-P P/P A/P 1 2 3 4 5 6 7 8 P/E 9 10 mRNA 250 5′ IB: ZAK (Strep) Phos-tag 150 3′ Collided 100 Stalled U4450 Kinase ZAK-P inactive ANS EDF1 U4450 C4398 C4398 250 IB: ZAK (Strep) Phos-tag 150 U4452 100 (kDa) c U4452 d RIM(c) SAM(c) RACK1(c) SAM(s) RIM(c) RACK1(s) RIH(c) RACK1(s) RACK1(c) mRNA RIH(c) RIH(s) RIH(s)-p mRNA RIH(s) RIH(s)-p EDF1 eS27(c) eS27(s) 60S pin(s) eS27(s) 60S 40S Collided Stalled Collided LZ P P SAM 277 339 416 RIM FPPLIK 422 417 Fig. 1 | Cryo-EM structure of ZAK bound to a colliding disome. a, Polysome profile (top) and immunoblots of a Phos-tag gel (IBs; bottom) from sucrose gradient fractions of HEK293T cells transfected with Strep-tagged WT and kinase inactive ZAK(T161A/S165A) expressed from a complete CMV promoter plasmid. Polysome profile from WT transfection is shown. b, Cut top view on the ZAK–disome model shown as low-pass-filtered surface, highlighting mRNA, tRNA and EDF1-binding sites. The boxed panels show a zoomed-in view on the ANS (red)-binding site of refined cryo-EM maps (transparent) of th (...truncated)