The Pro-apoptotic STK38 Kinase Is a New Beclin1 Partner Positively Regulating Autophagy

Abstract

SummaryAutophagy plays key roles in development, oncogenesis, cardiovascular, metabolic, and neurodegenerative diseases. Hence, understanding how autophagy is regulated can reveal opportunities to modify autophagy in a disease-relevant manner. Ideally, one would want to functionally define autophagy regulators whose enzymatic activity can potentially be modulated. Here, we describe the STK38 protein kinase (also termed NDR1) as a conserved regulator of autophagy. Using STK38 as bait in yeast-two-hybrid screens, we discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological, and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Upon autophagy induction, STK38-depleted cells display impaired LC3B-II conversion; reduced ATG14L, ATG12, and WIPI-1 puncta formation; and significantly decreased Vps34 activity, as judged by PI3P formation. Furthermore, we observed that STK38 supports the interaction of the exocyst component Exo84 with Beclin1 and RalB, which is required to initiate autophagosome formation. Upon studying the activation of STK38 during autophagy induction, we found that STK38 is stimulated in a MOB1- and exocyst-dependent manner. In contrast, RalB depletion triggers hyperactivation of STK38, resulting in STK38-dependent apoptosis under prolonged autophagy conditions. Together, our data establish STK38 as a conserved regulator of autophagy in human cells and flies. We also provide evidence demonstrating that STK38 and RalB assist the coordination between autophagic and apoptotic events upon autophagy induction, hence further proposing a role for STK38 in determining cellular fate in response to autophagic conditions.

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

“…Depletion of Beclin1, a key regulator of autophagy [42], also caused increased anoikis (Supplementary Figure S7) and interfered with anchorage-independent growth of HK-HRasG12V (Supplementary Figure S4). Considering that Beclin1 can function upstream of STK38 upon autophagy induction [29], these observations further strengthen the notion that STK38-mediated detachment-induced autophagy is critical to support oncogenic Ras signalling in order to antagonise anoikis and subsequently to promote anchorage-independent growth. In full agreement with this concept, we also detected considerably elevated STK38 kinase activity upon detachment of HK-HRasG12V cells (Figure 4M, 4N), as assessed by Thr444 phosphorylation of STK38, an established readout for STK38 kinase activity [27].…”

“…However, to our surprise, we found that STK38 supports the survival of Ras-transformed human cells upon cell detachment. Given this observation and that our research recently revealed that STK38 positively promotes autophagy in adherent cells [29], we therefore postulated that STK38-mediated autophagy might support anoikis resistance and/or anchorage-independent growth in Ras-transformed cells. Indeed, STK38 knockdown resulted in diminished detachment-induced autophagy and anchorage-independent growth, paralleled by increased anoikis.…”

“…Considering that detachment-induced autophagy can promote anchorage-independent growth of Ras-transformed cells [4], we explored whether the role of STK38 as autophagy regulator [29] might contribute to anchorage-independent growth of Ras-transformed human cells. Similar as reported for HCT116, Panc1 and H1299 [9, 13, 20], we observed that HK-HRasG12V displayed elevated detachment-induced autophagy (Figure 4A, 4B, Supplementary Figure S2D, S2E) as assessed by conversion of LC3B-I to LC3B-II [41].…”

“…For example, mitophagy maintains the pool of functional mitochondria necessary to support growth of Ras-driven tumours [13, 24], while Ras-induced autophagy can mediate the functional loss of mitochondria during transformation [25]. In this regard, it is intriguing that STK38 can act in mitochondrial quality control [45] in addition to its role in non-selective autophagy [29]. However, whether the STK38-mediated mitochondrial quality control pathway plays any role in cellular transformation is currently unknown.…”

“…Immunofluorescence (IF) and Western blot analyses were performed as defined [29]. IF images were acquired using a Zeiss Axiovert 200M Apotome microscope.…”