Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1

Abstract

BECN1/Beclin 1 is a critical protein in the initiation of autophagosome formation. Recent studies have shown that phosphorylation of BECN1 by STK4/MST1 at threonine 108 (T108) within its BH3 domain blocks macroautophagy/autophagy by increasing BECN1 affinity for its negative regulators, the antiapoptotic proteins BCL2/Bcl-2 and BCL2L1/Bcl-x L . It was proposed that this increased binding is due to formation of an electrostatic interaction with a conserved histidine residue on the anti-apoptotic molecules. Here, we performed biophysical studies which demonstrated that a peptide corresponding to the BECN1 BH3 domain in which T108 is phosphorylated (p-T108) does show increased affinity for anti-apoptotic proteins that is significant, though only minor (<2-fold). We also determined X-ray crystal structures of BCL2 and BCL2L1 with T108-modified BECN1 BH3 peptides, but only showed evidence of an interaction between the BH3 peptide and the conserved histidine residue when the histidine flexibility was restrained due to crystal contacts. These data, together with molecular dynamics studies, indicate that the histidine is highly flexible, even when complexed with BECN1 BH3. Binding studies also showed that detergent can increase the affinity of the interaction. Although this increase was similar for both the phosphorylated and non-phosphorylated peptides, it suggests factors such as membranes could impact on the interaction between BECN1 and BCL2 proteins, and therefore, on the regulation of autophagy. Hence, we propose that phosphorylation of BECN1 by STK4/MST1 can increase the affinity of the interaction between BECN1 and anti-apoptotic proteins and this interaction can be stabilized by local environmental factors.

Rac1 conditional deletion attenuates retinal ganglion cell apoptosis by accelerating autophagic flux in a mouse model of chronic ocular hypertension

“…Beclin1 is an autophagy effector that was originally identified as a Bcl-2-interacting protein. The pro-apoptotic factor Bak and anti-apoptotic factor Bcl-2 are both BH3-only members 50 – 52 , and the relative activities of BH3 proteins and initiator and/or executioner caspases decides that Beclin1 could either promote or inhibit autophagy 39 . Our co-IP results demonstrated an interaction between Beclin1 and Bak or Bcl-2 in both wt and Rac1 cKO mice.…”

Diversity in the intrinsic apoptosis pathway of nematodes

“…Affinity measurements by MST were performed using a Monolith NT.115 instrument (NanoTemper Technologies), as described previously 34 , employing purified, recombinant T. suis A ΔC28, T. suis B ΔC26 and BCL-XLΔC24 labelled using the NHS RED NanoTemper labelling kit (cat. no.…”

“…Pro-apoptotic BCL-2 proteins interact with pro-survival proteins via their BH3 domain. Hence, to further investigate whether the T. suis proteins A or B proteins interact, we purified recombinant proteins and measured their affinity for synthetic peptides corresponding either to their own BH3 domain, or that from the other protein, using microscale thermophoresis (MST), as previously 34 . No obvious interaction (K D > 50 μM) was observed between either protein and the others' BH3 domain, or its own BH3 sequence, suggesting that these proteins do not regulate each other as seen for mammalian BCL-2 proteins ( Supplementary Fig.…”

Emerging role of the Hippo pathway in autophagy

“…STK4/MST1 phosphorylates Beclin1 in its BH3 domain at Thr108, thereby inhibiting the Beclin1-Vps34 complex, which directly inhibits autophagy. Phosphorylation cascade can enhance Beclin1-Bcl-2 interaction and induce apoptosis [44][45][46] . RASSF1A, a Hippo pathway scaffold protein, binds to MST1, promotes the activation of MST1 and causes apoptosis (induced by the death receptor signaling pathway) 47,48 .…”