Stress-induced phosphorylation of CLIP-170 by JNK promotes microtubule rescue

Abstract

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170’s capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.

A liquid +TIP-network drives microtubule dynamics through tubulin condensation

“…Our in vitro results that nanomolar concentrations of EB3 and FL-CLIP undergo LLPS prompt the possibility that cellular +TIP-networks could be conducive to LLPS. To this end, we overexpressed GFP-CLIP-170 in live RPE-1 cells to analyze the characteristic plus-end "comet"-shaped profile (Perez et al, 1999;Henrie et al, 2020). We frequently saw trailing remnants that split off from the comets in a process reminiscent of droplet fission, and the remnants dissolved over time (Figure 5C and Movie 8).…”

Phase separation of +TIP-networks regulates microtubule dynamics

“…Overexpressed EB3 and CLIP exhibit atypical network formation at the growing microtubule tip, leaving trailing protein "remnants" behind the leading network edge (Mustyatsa et al, 2019;Henrie et al, 2020). One explanation for these remnants is that they form by binding to tubulin "islands" remaining in the GTP-state behind the GTP-cap (Perez et al, 1999;de Forges et al, 2016;Henrie et al, 2020).…”

Cytoskeleton and Associated Proteins: Pleiotropic JNK Substrates and Regulators

“…In epithelial cells, JNK1 and JNK2 can directly phosphorylate the MT-rescue factor CLIP-170 at Thr25, Thr45 and Ser147 with different kinetics and efficacies upon stress [122]. These phosphorylations located in serine-rich regions flank the CAP-Gly MT-binding and SxIP EB1-binding domains and allow frequent formation of short-lived CLIP-170 remnants at the trailing end of comets.…”

“…Interestingly, MAP4, HDAC6 and dynein/dynactin, which are both MAPs and SeptAPs, all contain SP/TP sites which are phosphorylated in growth cones of rat neurons, suggesting that they may be substrates of JNK at that location [70] (Section 6 and Supplementary Table S1). CLIP-170 is also a known substrate of JNK regulating MT rescues [122]. Lastly, note that JIPs might be involved in septin transport, since endosomal trafficking of septin mRNAs, and their local translation and assembly in heteromeric complexes on MTs, is required in fungi to promote efficient septin cytoskeleton formation at appropriate location [182].…”