Identification of Kinases and Phosphatases That Regulate ATG4B Activity by siRNA and Small Molecule Screening in Cells.

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

Phosphatidylinositol 4-kinase IIß negatively regulates invadopodia formation and suppresses an invasive cellular phenotype.

The type II phosphatidylinositol 4-kinase (PI4KII) enzymes synthesize the lipid phosphatidylinositol 4-phosphate (PI(4)P), which has been detected at the Golgi complex and endosomal compartments and recruits clathrin adaptors. Despite common mechanistic similarities between the isoforms, the extent of their redundancy is unclear. We found that depletion of PI4KIIα and PI4KIIß using small interfering RNA led to actin remodeling. Depletion of PI4KIIß also induced the formation of invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). Depletion of PI4KII isoforms also differentially affected trans-Golgi network (TGN) pools of PI(4)P and post-TGN traffic. PI4KIIß depletion caused increased MT1-MMP trafficking to invasive structures at the plasma membrane and was accompanied by reduced colocalization of MT1-MMP with membranes containing the endosomal markers Rab5 and Rab7 but increased localization with the exocytic Rab8. Depletion of PI4KIIß was sufficient to confer an aggressive invasive phenotype on minimally invasive HeLa and MCF-7 cell lines. Mining oncogenomic databases revealed that loss of the PI4K2B allele and underexpression of PI4KIIß mRNA are associated with human cancers. This finding supports the cell data and suggests that PI4KIIß may be a clinically significant suppressor of invasion. We propose that PI4KIIß synthesizes a pool of PI(4)P that maintains MT1-MMP traffic in the degradative pathway and suppresses the formation of invadopodia.

EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src.

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

Loss of INPP4B causes a DNA repair defect through loss of BRCA1, ATM and ATR and can be targeted with PARP inhibitor treatment.

Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.

Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr577 subsequent to its Src-mediated phosphorylation at Tyr576. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹89 and Tyr¹¹9°, as well as Tyr¹¹85, and downstream phosphorylation of the kinase AKT at Thr³°8 were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser47³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.