Masatoshi Maki

Cell death-inducing p53-target protein 1 (CDIP1) is a proapoptotic protein that is normally expressed at low levels and is upregulated by genotoxic and endoplasmic reticulum stresses. CDIP1 has been reported to be localized to endosomes and to interact with several proteins, including B-cell receptor-associated protein 31 (BAP31) and apoptosis-linked gene 2 (ALG-2). However, the cellular and molecular mechanisms underlying CDIP1 expression-induced apoptosis remain unclear. In this study, we first demonstrated that CDIP1 was upregulated after treatment with the anticancer drug adriamycin in human breast cancer MCF-7 cells but was degraded rapidly in the lysosomal pathway. We also demonstrated that treatment with the cyclin-dependent kinase 5 (CDK5) inhibitor roscovitine led to an increase in the electrophoretic mobility of CDIP1. In addition, a phosphomimetic mutation at Ser-32 in CDIP1 resulted in an increase in CDIP1 expression-induced apoptosis. We also found that CDIP1 expression led to the induction of autophagy prior to apoptosis. Treatment of cells expressing CDIP1 with SAR405, an inhibitor of the class III phosphatidylinositol 3-kinase VPS34, caused a reduction in autophagy and promoted apoptosis. Therefore, autophagy is thought to be a defense mechanism against CDIP1 expression-induced apoptosis.

Environmental factors are important regulators of cell growth and proliferation. Mechanistic target of rapamycin (mTOR) is a central kinase that maintains cellular homeostasis in response to a variety of extracellular and intracellular inputs. Dysregulation of mTOR signaling is associated with many diseases, including diabetes and cancer. Calcium ion (Ca²⁺) is important as a second messenger in various biological processes, and its intracellular concentration is tightly regulated. Although the involvement of Ca²⁺ mobilization in mTOR signaling has been reported, the detailed molecular mechanisms by which mTOR signaling is regulated are not fully understood. The link between Ca²⁺ homeostasis and mTOR activation in pathological hypertrophy has heightened the importance in understanding Ca²⁺-regulated mTOR signaling as a key mechanism of mTOR regulation. In this review, we introduce recent findings on the molecular mechanisms of regulation of mTOR signaling by Ca²⁺-binding proteins, particularly calmodulin (CaM).

Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca²⁺/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca²⁺ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca²⁺/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca²⁺/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.

Apoptosis-linked gene 2 (ALG-2, also known as PDCD6) is a member of the penta-EF-hand (PEF) family of Ca²⁺-binding proteins. The murine gene encoding ALG-2 was originally reported to be an essential gene for apoptosis. However, the role of ALG-2 in cell death pathways has remained elusive. In the present study, we found that cell death-inducing p53 target protein 1 (CDIP1), a pro-apoptotic protein, interacts with ALG-2 in a Ca²⁺-dependent manner. Co-immunoprecipitation analysis of GFP-fused CDIP1 (GFP-CDIP1) revealed that GFP-CDIP1 associates with tumor susceptibility gene 101 (TSG101), a known target of ALG-2 and a subunit of endosomal sorting complex required for transport-I (ESCRT-I). ESCRT-I is a heterotetrameric complex composed of TSG101, VPS28, VPS37 and MVB12/UBAP1. Of diverse ESCRT-I species originating from four VPS37 isoforms (A, B, C, and D), CDIP1 preferentially associates with ESCRT-I containing VPS37B or VPS37C in part through the adaptor function of ALG-2. Overexpression of GFP-CDIP1 in HEK293 cells caused caspase-3/7-mediated cell death. In addition, the cell death was enhanced by co-expression of ALG-2 and ESCRT-I, indicating that ALG-2 likely promotes CDIP1-induced cell death by promoting the association between CDIP1 and ESCRT-I. We also found that CDIP1 binds to vesicle-associated membrane protein-associated protein (VAP)A and VAPB through the two phenylalanines in an acidic tract (FFAT)-like motif in the C-terminal region of CDIP1, mutations of which resulted in reduction of CDIP1-induced cell death. Therefore, our findings suggest that different expression levels of ALG-2, ESCRT-I subunits, VAPA and VAPB may have an impact on sensitivity of anticancer drugs associated with CDIP1 expression.

ALG-2 is a penta-EF-hand Ca²⁺-binding protein and interacts with a variety of proteins in mammalian cells. In order to find new ALG-2-binding partners, we searched a human protein database and retrieved sequences containing the previously identified ALG-2-binding motif type 2 (ABM-2). After selecting 12 high-scored sequences, we expressed partial or full-length GFP-fused proteins in HEK293 cells and performed a semi-quantitative in vitro binding assay. SARAF, a negative regulator of store-operated Ca²⁺ entry (SOCE), showed the strongest binding activity. Biochemical analysis of Strep-tagged and GFP-fused SARAF proteins revealed ubiquitination that proceeded during pulldown assays under certain buffer conditions. Overexpression of ALG-2 interfered with ubiquitination of wild-type SARAF but not ubiquitination of the F228S mutant that had impaired ALG-2-binding activity. The SARAF cytosolic domain (CytD) contains two PPXY motifs targeted by the WW domains of NEDD4 family E3 ubiquitin ligases. The PPXY motif proximal to the ABM-2 sequence was found to be more important for both in-cell ubiquitination and post-cell lysis ubiquitination. A ubiquitination-defective mutant of SARAF with Lys-to-Arg substitutions in the CytD showed a slower degradation rate by half-life analysis. ALG-2 promoted Ca²⁺-dependent CytD-to-CytD interactions of SARAF. The ALG-2 dimer may modulate the stability of SARAF by sterically blocking ubiquitination and by bridging SARAF molecules at the CytDs.

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca²⁺ elevation. However, Ca²⁺ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca²⁺-calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca²⁺-mobilizing agonists independent of AP-1 activation.

Many non-catalytic cellular proteins exert biological functions by formation of stable or transient complexes with other proteins. Analysis of the signal-induced physical interactions is important to understand their physiological roles in cells. Here we describe a biochemical method for assessing the binding of ALG-2 (gene name, PDCD6) to its target proteins that are immunoprecipitated from cell lysates. Application of nanoluciferase (Nluc)-fused ALG-2 enables a rapid quantitative evaluation of Ca²⁺-dependent interactions of target proteins with ALG-2 in vitro binding assays.

NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca²⁺/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca²⁺-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca²⁺-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca²⁺-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca²⁺-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca²⁺ mobilization.