The Autophagy-Initiating Kinase ULK1 Controls RIPK1-Mediated Cell Death.

Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death.

Phosphorylation of Syntaxin 17 by TBK1 Controls Autophagy Initiation.

Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13+FIP200+ mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17pS202 transfers from the Golgi, where its steady-state pools localize, to the ATG13+FIP200+ mPAS. Stx17pS202 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17S202A was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17pS202 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.

Inhibition of protein phosphatase 2A (PP2A) prevents Mcl-1 protein dephosphorylation at the Thr-163/Ser-159 phosphodegron, dramatically reducing expression in Mcl-1-amplified lymphoma cells.

Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells.