PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase-3/6/7 regulates heat-induced activation in oysters

Wang, Chaogang; Du, Mingyang; Jiang, Zhuxiang; Cong, Rihao; Wang, Wei; Zhang, Taiping; Chen, Jincheng; Zhang, Guofan; Li, Li

doi:10.1038/s42003-024-07184-4

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Published: 07 November 2024

PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase-3/6/7 regulates heat-induced activation in oysters

Chaogang Wang^1,2,3,4^na1,
Mingyang Du^3,4,5^na1,
Zhuxiang Jiang^3,4,5,
Rihao Cong^2,3,4,6,
Wei Wang^3,4,7,
Taiping Zhang^3,4,5,
Jincheng Chen^3,4,5,
Guofan Zhang^1,2,3,4,6,8 &
…
Li Li ORCID: orcid.org/0000-0002-5913-4447^1,3,4,5,7,8

Communications Biology volume 7, Article number: 1459 (2024) Cite this article

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Abstract

Cysteine-aspartic proteases (caspases) are critical drivers of apoptosis, exhibiting expansion and domain shuffling in mollusks. However, the functions and regulatory mechanisms of these caspases remain unclear. In this study, we identified a group of Caspase-3/6/7 in Bivalvia and Gastropoda with a long inter-subunit linker (IL) that inhibits cleavage activation. Within this region, we found that conserved phosphorylation at Thr260 in oysters, mediated by the PI3K-AKT pathway, suppresses heat-induced activation. This mechanism is involved in divergent temperature adaptation between two allopatric congeneric oyster species, the relatively cold-adapted Crassostrea gigas and warm-adapted Crassostrea angulata. Our study elucidates the role of these effector caspase members and their long IL in bivalves, revealing that the PI3K-AKT pathway phosphorylates Thr260 on CgCASP3/6/7’s linker to inhibit heat-induced activation. These findings provide insights into the evolution and function of apoptotic regulatory mechanisms in bivalves.

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Introduction

Cell apoptosis is a physiological protective mechanism that plays a crucial role in responding to biotic and abiotic stresses, as well as in developing and maintaining organismal homeostasis¹. It selectively eliminates redundant, damaged or potentially harmful cells from the body^2,3. Apoptosis involves characteristic morphological changes driven by cysteine-aspartic proteases (caspases), typically initiated when initiator caspases (caspase-2, -8, -9, and -10) receive apoptotic signals. These initiator caspases then cleave and activate effector caspases (caspase-3, -6, and -7), which contain a conserved cysteine active site (QACXG) and are composed of large (p20) and small (p10) subunits, to execute the final steps of apoptosis⁴.

Mollusks, the second-largest phylum in the animal kingdom, exhibit a complex process of apoptosis⁵. The increasing availability of whole-genome and transcriptome data has greatly enhanced our understanding of the diverse functions and evolution of caspases in these organisms⁶. For example, the genome of the Pacific oyster, Crassostrea gigas, reveals a significant expansion of the caspase gene family, possibly due to its aquatic lifestyle and limited mobility-induced exposure to various pathogens, balanced by the expansion of inhibitors of apoptosis proteins (IAPs)⁷. Unlike many other organisms, oysters lack direct homologs of essential caspases, such as caspase-9, -1, -3, -6, and -7^6,8. Instead, they possess novel members of the caspase family with unique motifs within their prodomains, such as caspase-3/7, highlighting the unique caspase functions and regulatory mechanisms in mollusks⁶.

Several studies have identified homologs of the caspase family in various mollusk species, including initiator and effector caspases^9,10,11, reported new effector caspases with domain shuffling (gene fragment recombination leads to the recombination and rearrangement of protein domains)¹², and explored their expression in response to various stresses and at different developmental stages^13,14. However, the specific functions, upstream regulatory mechanisms, and unique domains of these novel members still remain unclear.

Protein phosphorylation, a critical post-translational modification, plays a significant role in cellular apoptosis^15,16,17. The addition or removal of phosphate groups at specific protein sites can cause substantial biochemical changes, influencing signaling pathways and protein conformations^18,19. Caspases, a subset of kinase substrates, are directly affected by phosphorylation in model organisms²⁰. For example, caspase-9 has the most phosphorylation sites among caspases, which are distributed across its structural domains and impact cleavage, dimerization, protein-protein interactions, and apoptotic body formation²¹. However, research on the phosphorylation of effector caspases is still limited, even in model organisms. Studies have shown that CK2²², AMPK²³ and PAK2²⁴ phosphorylate caspase-3 (Thr174 and Ser176), caspase-6 (Ser257), and caspase-7 (Ser30 and Ser239) to inhibit their activation in humans. Currently, there are no reports on the phosphorylation of caspase in mollusks, which possess unique and complex caspases.

Oysters are widely distributed model mollusks with early deciphered genomes, and they hold significant economic and ecological value⁷. Without an adaptive immune system, oysters have developed a complex apoptotic system to cope with drastic fluctuations in temperature, salinity, and nutrients, as well as pathogen invasion in estuarine and intertidal regions²⁵. C. gigas and Crassostrea angulata are two allopatric congeneric oyster species adapted to different thermal environments along the northern and southern coasts of China, respectively, resulting in higher thermal tolerance in C. angulata^{7,26,27,28,29}. Our previous study found that C. angulata, which shows lower apoptosis rates under heat stress, had higher phosphorylation levels at Thr260 in the novel effector CASP protein compared to that of C. gigas under heat stress³⁰. This suggests that phosphorylation modifications may regulate the enzymatic activity of oysters’ novel caspase-3/6/7 under heat stress, contributing to divergent temperature adaptations. Comparative studies of these species should help to characterize the function of novel caspase members and elucidate the molecular mechanisms of their activation through phosphorylation.

In this study, we identified a group of novel Caspase-3/6/7 proteins in Bivalvia and Gastropoda that possess a long inter-subunit linker (IL) capable of inhibiting activation. Phylogenetic analysis and biochemical experiments revealed that the oysters’ conserved phosphorylation modification site Thr260, within the IL of CgCaspase-3/6/7 suppresses heat-induced activation and is directly phosphorylated by the phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)-AKT pathway. This study characterizes the function of these novel caspase members and their unique IL in bivalves, reporting for the first time that phosphorylation of the IL in oysters inhibits caspase activation via the PI3K-AKT pathway. These findings reveal the existence of complex and unique apoptotic regulatory mechanisms in bivalves and provide new insights into the evolution and function of the crosstalk between the PI3K-AKT and caspase pathways.

Results

Bivalvia and Gastropoda have evolved novel caspase-3/6/7 genes with long IL

To investigate the compositional differences in effector caspases gene across metazoans, we annotated 187 homologs of human caspases (HmCaspase-3, HmCaspase-6, and HmCaspase-7) in the genomes of 21 representative species from diverse evolutionary backgrounds (Supplementary Fig. 1; Supplementary Data 1). In addition to model vertebrates, such as Homo sapiens, Mus musculus and Danio rerio, these homologs generally fell into three clusters: Caspase-6 like, Caspase-3/7 like and Caspase-3/6/7 like (Fig. 1). The lengths of the N-terminal, P20, IL, P10, and C-terminal domains varied among these groups. Notably, unlike the short IL (<60 amino acids [AA]) identified in most effector caspases of other species, we discovered a distinct cluster of caspase-3/6/7 in Bivalvia and Gastropoda with a long IL (63–286 AA) (Fig. 1; Supplementary Data 1). Pairwise comparisons showed low similarity among these long ILs (Supplementary Fig. 2). Collinearity analysis of the C. gigas genome revealed that the novel caspase-3/6/7 genes likely arose from tandem duplications (Supplementary Fig. 3; Supplementary Table 1) and intraexonic insertion/deletion due to a lack of homology between exons and introns (Supplementary Fig. 4). Gene expression analysis showed that these novel caspase-3/6/7 genes were highly expressed in gills, labial palps and hemocytes, varied across developmental stages, and responded to stressors like temperature, salinity, and heavy metal (Supplementary Fig. 5). Taking Cg CASP3/6/7 like6 (referred to as CgCASP3 hereafter) as an example, the sequence alignment indicated that it has a P20 domain (54V–201S), a long IL (202N–366M), a short p10 domain (422A–P515), and a conserved caspase family cysteine active-site motif, QACRS (Supplementary Fig. 6).

**Fig. 1: Phylogenetic tree of effector *Caspase* genes from representative animals across metazoans.**

The long IL in CgCASP3 inhibited its cleavage activation

In vitro substrate specificity assays were performed to investigate the function of the CgCASP3. The results showed that purified CgCASP3 specifically catalyzed the cleavage of CASP3 and the CASP7 substrate Ac-DEVD-pNA (Fig. 2A). This cleavage activity was significantly inhibited by the pan-caspase inhibitor Z-VAD-FMK and the caspase-3/-7 inhibitor Ac-DEVD-CHO (Fig. 2B). A Coomassie brilliant blue stain revealed that CgCASP3-∆M, a deletion mutant lacking IL, was more susceptible to PAC-1-induced self-cleavage compared to wild-type CgCASP3 (Fig. 2C). Subsequently, we conducted apoptosis activation experiments following in vivo cell transfection to assess the role of the long IL in regulating CgCASP3-mediated apoptosis. The cell-based transfection DEVD-ase activity assay showed that with the increasing treatment time of the apoptosis inducer (TNF-α + SM-164), cells transfected with CgCASP3 exhibited significantly higher DEVD-ase activity compared to those transfected with the empty plasmid pCMV-N-mCherry (control group). Furthermore, cells transfected with CgCASP3-∆M displayed the highest DEVD-ase activity, which was significantly higher than that of the CgCASP3 group at 8 h (p < 0.05; Fig. 2D). Similarly, the results from the CCK-8 (Cell Counting Kit-8) cell viability assay (Fig. 2E), LDH (lactate dehydrogenase) release (Fig. 2F), and cell apoptosis experiments (Fig. 2G) also showed the same trend, that under treatment of apoptosis inducer (TNF-α + SM-164), CgCASP3-∆M was more prone to triggering cellular apoptosis compared to CgCASP3 (p < 0.05). The TUNEL assay revealed that under treatment of apoptosis inducer, CgCASP3, indicated by red fluorescence, was distributed in the cytoplasm and nucleus, while CgCASP3-∆M significantly increased the number of apoptotic TUNEL+ cells (indicated by green fluorescence) compared to CgCASP3 (p < 0.001; Supplementary Fig. 7). The western blotting further confirmed that with increasing treatment time of the apoptosis inducer, the content of ProCgCASP3 decreased, while the cleaved PARP1 significantly increased compared to the control group. Moreover, the transfection of the CgCASP3-∆M group exhibited a faster decline/increase in ProCgCASP3/cleaved PARP1 content compared to the CgCASP3 group (Fig. 2H). The in vitro DEVD-ase activity assay also supported that CgCASP3-∆M had significantly higher activity than CgCASP3 (p < 0.05; Fig. 2I). The findings demonstrate that CgCASP3 possesses CASP-3/-7 enzymatic activity and that a long IL inhibits its cleavage activation.

**Fig. 2: The long IL of novel *CgCaspase-3/6/7* inhibits its cleavage activation.**

Phosphorylation of CgCASP3 at Thr260 inhibited its cleavage activation

Our previous experiments found that during heat stress, the phosphorylation levels at the Thr260 site of CgCASP3/6/7 like6 increased by 1.4-fold in C. gigas (which has relatively lower thermal tolerance) and decreased by 0.5-fold in C. angulata (which has relatively higher thermal tolerance) during heat stress between (Fig. 3A)³⁰. To assess the effects of Thr260 phosphorylation, we measured DEVD-ase activity in the gill tissues of both species under heat stress. The results showed that high temperatures significantly increased DEVD-ase activity in both species, however, the activity was significantly lower in C. angulata compared to C. gigas after heat stress (p < 0.05; Fig. 3B). Sequence alignment indicated that the phosphorylation modification (Ser and Thr) at the Thr260 site of Cg CASP3/6/7 like6 was conserved in some of the oysters’ novel Caspase-3/6/7 genes (Fig. 3C). We then constructed plasmids with Thr260 site mutations (CgCASP3^T260A, mimicking dephosphorylation; CgCASP3^T260D, mimicking phosphorylation) and performed cell transfection and apoptosis induction experiments to assess the effects of Thr260 phosphorylation on apoptosis regulation. The results showed that with the increasing treatment time of the apoptosis inducer (TNF-α + SM-164), DEVDase activity (p < 0.005; Fig. 3D), LDH release (p < 0.05; Fig. 3F), and cell apoptosis rates (p < 0.05; Fig. 3G) were significantly higher in cells transfected with the CgCASP3^T260A mutant compared to those transfected with CgCASP3. In contrast, cells transfected with the CgCASP3^T260D mutant exhibited significantly lower levels of these indicators than the other two groups. CCK-8 cell viability assays showed the opposite trend (p < 0.005; Fig. 3E). Western blotting revealed that, with increasing apoptotic induction time, the rate of decrease in ProCgCASP3 content followed the order CgCASP3^T260A > CgCASP3 > CgCASP3^T260D, in contrast, the rate of increase in cleaved PARP1 content followed the opposite trend (Fig. 3H). The in vitro DEVD-ase activity assay also confirmed this order: CgCASP3^T260A > CgCASP3 > CgCASP3^T260D (Fig. 3I). These findings demonstrate that phosphorylation of Thr260 in CgCASP3 significantly inhibits its ability to induce apoptosis.

**Fig. 3: The phosphorylation of Thr260 at *CgCaspase-3/6/7* inhibits its activation.**

AKT phosphorylation of Thr260 in CgCASP3 inhibits heat-activation

The kinase prediction analysis for the Thr260 site of Cg CASP3/6/7 like6 showed that AKT kinase had the highest predicted score (Supplementary Table 2). We then used co-immunoprecipitation (Co-IP), yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and subcellular co-localization experiments to validate the interaction between CgAKT and CgCASP3. Co-IP results confirmed a specific interaction between CgCASP3 and CgAKT in cell lysates co-transfected with Flag-CgCasp3 and Myc-CgAkt (Fig. 4A). Yeast two-hybrid assays further validated this interaction (Fig. 4B). We fused CgCASP3 to the GAL4 DNA-binding domain and CgAKT to the GAL4 transcriptional activation domain. Yeast cells co-transformed with pGADT7-CgAKT and pGBKT7-CgCASP3 grew on SD/-Leu/-Trp/-His3 and SD/-Leu/-Trp/-His3/-Ade2 media, similar to the positive control (pGADT7-T + pGBKT7-53). In contrast, yeast co-transformed with empty vectors did not grow on selective media, consistent with the negative control (pGADT7-T + pGBKT7-lam) (Fig. 4B). BiFC analysis showed that cells co-transfected with pBiFC-VC155-CgAKT and pBiFC-VN173-CgCASP3 exhibited green fluorescence in the cytoplasm and nucleus, in contrast, no fluorescence was detected when either plasmid was co-transfected with an empty vector (Fig. 4C). Subcellular localization experiments indicated that under heat stress, CgAKT localized to cell membrane, suggesting activation, and co-localization fluorescence analysis confirmed their interaction (Fig. 4D). To further verify the kinase-substrate relationship between CgAKT and CgCASP3, we performed in vitro and in vivo kinase assays. The in vitro assay showed a well-distinguishable band with the anti-PS/PT antibody when CgAKT was co-incubated with CgCASP3, which disappeared when CgCASP3^T260A was used instead (Fig. 4E). In vivo kinase experiments demonstrated that CgAKT phosphorylated the T260 site of CgCASP3, indicated by a stronger anti-pS/pT band when CgAKT and CgCASP3 were co-transfected, compared to CgCASP3 alone. This phosphorylation was absent when CgCASP3^T260A was transfected, regardless of CgAKT co-transfection (Fig. 4F). We then explored the effects of CgAKT on CgCASP3-mediated apoptosis through phosphorylation by co-transfecting CgAKT with CgCASP3 mutants, and conducting apoptosis induction experiments. DEVD-ase activity assay (Fig. 4G), CCK-8 cell viability (Fig. 4H), LDH release (Fig. 4I), and cell apoptosis assay (Fig. 4J; Supplementary Fig. 8) demonstrated that CgAKT significantly inhibited CgCASP3-induced apoptosis, which CgCASP3^T260A reversed. Similarly, CgAKT co-transfection reduced ProCgCASP3 cleavage and cleaved PARP1 content, effects that were prevented by CgCASP3^T260A (Supplementary Fig. 9). Further experiments using the AKT inhibitor MK-2206 and SC79 (the AKT activator) SC79, in combination with CgCASP3 and CgAKT co-transfection, showed that the inhibiting or activation CgAKT decreased or increased CgCASP3 phosphorylation, facilitated or inhibited ProCgCASP3 cleavage, and modulated PARP1 levels (Supplementary Fig. 10; Supplementary Fig. 11). This modulation of CgAKT activity also affected DEVD-ase activity (Supplementary Fig. 12A), cell viability (Supplementary Fig. 12B), LDH release (Supplementary Fig. 12C), and apoptosis rate (Supplementary Fig. 12D) in cells transfected with CgCASP3. These results indicate that CgAKT phosphorylates Thr260 of CgCASP3 to inhibit its cleavage and suppress CgCASP3-mediated apoptosis.

**Fig. 4: CgAKT phosphorylates Thr260 of *CgCaspase-3/6/7* to inhibits its activation.**

PI3K-AKT signaling pathway mediated CgCASP3 Thr260 phosphorylation

Based on these results, we planned to investigate further the differences in the PI3K-AKT pathway between C. gigas and C. angulata under heat stress. During the PI3K-AKT pathway, phosphoinositide 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol 3,4,5-trisphosphate, which in turn activates downstream AKT kinase to phosphorylate numerous target proteins, thereby regulating cell survival, proliferation, growth, and metabolism. Sequence alignment revealed that the CgAKT residues Thr291 and Ser464 correspond to the conserved active sites Thr308 and Ser473 in HmAKT1 (Fig. 5A, Supplementary Fig. 13). Western blotting results showed that the phosphorylation levels of CgAKT^T291 (HmAKT1^T308) along with its upstream regulators in the PI3K-AKT pathway, including the protein content and phosphorylation levels (Ser241) of PDPK1 and the protein content of PIK3CA, were higher in C. angulata than C. gigas after heat stress. Conversely, the PTEN protein content was lower in C. angulata than in C. gigas following heat stress (Fig. 5B).

**Fig. 5: The phosphorylation of CgCASP3 Thr260 site is regulated by classic PI3K-AKT pathway.**

Due to the low homology between the sequences surrounding the S464 site of CgAKT and the S473 site of HmAKT1, we could not measure the phosphorylation levels of CgAKT^S464 in the two species by western blotting. However, based on the expression (Supplementary Fig. 14) and phosphorylation levels (Supplementary Fig. 15) of mTOR (a kinase involved in phosphorylating CgAKT at the Ser464 site) from previous studies, it was suggested that the phosphorylation level of the S464 site of CgAKT might be higher in C. angulata than in C. gigas.

Moreover, our previous transcriptomic data from C. gigas and C. angulata under heat stress revealed that the expression level of the phosphatase PP2AC, which mediates dephosphorylation of Thr291 and Ser464 sites in CgAKT, was significantly downregulated in C. angulata and lower than in C. gigas after heat stress (p < 0.001; Supplementary Fig. 16).

Both in vivo kinase assay (Fig. 5C) and in vitro kinase experiment (Fig. 5D) with CgAKT^{T291D S464D} mutant (mimicking phosphorylation) and CgAKT^{T291A S464A} mutant (mimicking dephosphorylation) provided compelling evidence that phosphorylation at CgAKT T291 and S464 significantly enhanced kinase activity, leading to an elevated phosphorylation level of CgCASP3 T260 site. Western blotting results of cell apoptosis similarly demonstrated that simulating CgAKT phosphorylation/dephosphorylation to activate/deactivate its kinase activity can significantly inhibited/promoted ProCgCASP3 cleavage and decreased/increased cleaved PARP1 (Fig. 5E). Further cell-based transfection assays for DEVD-ase activity (p < 0.05; Fig. 5F), CCK-8 cell viability (p < 0.05; Fig. 5G), LDH release (p < 0.05; Fig. 5H), and cell apoptosis rates (p < 0.05; Fig. 5I; Supplementary Fig. 17) demonstrated that AKT phosphorylation at T291 and S464 directly influenced CgCASP3 cleavage activation by modulating kinase activity.

To explore the impact of upstream regulatory factors in the PI3K-AKT pathway, mediating CgAKT T291 phosphorylation modification and the corresponding CgCASP3 activity, we conducted co-transfection experiments combined with in vivo kinase assays (Fig. 5J), cell apoptosis western blotting (Fig. 5K), cell-based transfection DEVD-ase activity assay (p < 0.05; Supplementary Fig. 18A), CCK-8 cell viability assays (p < 0.01; Supplementary Fig. 18B), LDH release measurements (p < 0.05; Supplementary Fig. 18C), and cell apoptosis experiments (p < 0.05; Supplementary Fig. 18D). The results revealed that CgPDPK1 enhanced CgAKT kinase activity by phosphorylating its T291 site, which then phosphorylated CgCASP3 T260 site to inhibit cleavage activation and CgCASP3-induced cell apoptosis. This effect was amplified by CgPIK3CA and suppressed by CgPTEN.

These results suggest that the PI3K-AKT pathway exhibits a stronger activation pattern in C. angulata during heat stress, which inhibits CgCASP3 cleavage by enhancing Thr260 phosphorylation, ultimately contributing to the lower heat-induced mortality of C. angulata.

Discussion

Apoptosis is a regulated form of cell death characterized by specific morphological features and is dependent on caspases, occurring during metazoan development, tissue homeostasis, and regeneration^31,32. In this study, we analyzed homologs of effector caspase genes in both vertebrates and invertebrates. The results showed that, in addition to well-studied model organisms (Homo sapiens, Mus musculus and Danio rerio), effector caspase genes in other species were classified into three clusters (CASP3/7, CASP6, and CASP3/6/7) and exhibited variations in member numbers and the length of the N-terminal, P20, IL, P10, and C-terminal domains. Notably, mollusks exhibited significant gene expansion, and bivalves and gastropods specifically evolved a set of caspase-3/6/7 genes with long IL regions, similar to the previously identified CgCASP-3/7 in C. gigas; however, they have been assigned different names¹². Previous studies have shown that bivalves possess a complex caspase family with expanded effector caspases that do not have direct homologs in vertebrates⁶. These caspases form specific clusters that may be species-specific in some cases. For example, Cg CASP3/6/7 like6 encodes a protein consisting of 464 AA with a short P10 domain (44 AA), a long IL (165 AA), and a mutated cysteine active-site motif QACRS, which deviates from the highly conserved cysteine active site QACRG motif³³. This mutated cysteine active-site motif can also be observed in caspase-3/7-3 of Mytilus galloprovincialis⁹. Functional experimental results demonstrated that the long IL significantly inhibited the cleavage activation of CgCASP3, which regulates cysteine protease activity.

Studies on CASP3 and CASP7 in vertebrates have reported that effector caspases exist as inactive dimers, with a short IL region binding to the dimer interface, preventing the formation of the substrate-binding pocket, which is cleaved during maturation⁴. The long IL of CgCASP3 proteins may hinder the formation of a substrate-binding pocket by increasing the spatial distance of the dimer interface, which is involved in the precise regulation and efficient apoptotic responses of significantly expanded caspases in bivalves and gastropods encountering various biotic and abiotic stressors. Subsequent analysis of the oyster genome revealed a slight expansion of caspase-3/6/7 genes in mollusks, originating from tandem and segmental duplications. This observation aligns with similar findings of gene expansion within apoptosis-related gene families, such as IAP, in other mollusk genomes^7,34.

However, given the low similarity between the long IL of the novel Caspase-3/6/7 genes in bivalves and gastropods, these findings may not apply to all genes within this cluster. Furthermore, the novel caspase-3/7 genes exhibited higher expression levels in the gills, labial palps, and hemocytes, suggesting a role in stress resistance and pathogen recognition and elimination in response to various abiotic and biotic stressors^35,36. Additionally, these genes displayed divergent expression patterns during development, as seen with Cg CASP3/6/7 like6, which showed elevated expression from the egg to the morula stage. This suggest function in shaping and refining tissues and organs during embryonic development^37,38.

Caspase regulation is crucial for determining cells survival or death. It has been demonstrated that the phosphorylation of initiator and effector caspases often leads to their inactivation²¹. In this study, we reported for the first time that the conserved phosphorylation at Thr260 within the long IL of CgCASP3 inhibited its cleavage and activation. As previously reported in many model organisms, phosphorylation at the IL can directly block the cleavage of several caspases, such as caspase-2³⁹, caspase-3²², caspase-7²⁴, caspase-8^40,41, and caspase-9⁴². The negative charge of the phosphorylated residues in the linker region can distort the three-dimensional structural interface near the dimer, disrupting inter-monomer interactions and thereby impeding caspase cleavage and activation⁴³.

Kinase prediction and kinase-substrate experiments confirmed that CgAKT act as the upstream kinase responsible for phosphorylating the Thr260 site of CgCASP3. AKT, also known as PKB or Rac, is crucial in regulating cell survival and apoptosis^44,45, and can be activated by high temperatures and oxidative stress^46,47. Previous research demonstrated that AKT phosphorylates the Ser196 site of HmCASP9, thereby inhibiting its proteolytic activity⁴⁸. Additionally, studies exploring the crosstalk between the AKT and caspase pathways, particularly with effector caspases, have generally observed a negative correlation between HmAKT1 phosphorylation levels and the activity of HmCASP3 or HmCASP7^49,50,51.

Phosphorylation of the Thr308 (corresponding to CgAKT^T291) and Ser473 (corresponding to CgAKT^S464) sites is required for the full activity of AKT1⁵². T308 is phosphorylated by PDPK1⁵³, in contrast, Ser473 is phosphorylated by mTOR⁵⁴. Both sites are dephosphorylated and inactivated by PP2A⁵⁴. By comparing previous data and western blot results from two differential heat-tolerant oyster species, C. gigas and C. angulata, during heat stress, we observed an upregulation of phosphorylation at the critical AKT residue T291 in C. angulata; in contrast, C. gigas showed a significant downregulation. The upstream regulators involved in the phosphorylation of this site, including the phosphorylation level of S241 at PDPK1 and the protein levels of PTEN and PIK3CA, showed a similar trend. The Ser241 site within the activation loop of PDPK1 is crucial for activating its kinase domain as it undergoes autophosphorylation to initiate self-activation⁵⁵. As a dual-specificity protein phosphatase, PTEN counteracts the PI3K-AKT signaling pathway by dephosphorylating phosphoinositides⁵⁶. PIK3CA, the critical catalytic subunit of PI3K, utilizes ATP and PtdIns (4,5) P2 (phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which recruits proteins containing PH domains, such as AKT1 and PDPK1, to the membrane, thereby activating downstream AKT kinases^57,58.

The PI3K-AKT pathway is widely recognized to be activated by high temperatures, which helps counteract oxidative stress, promotes energy metabolism, stimulates cell proliferation and growth, and inhibits apoptosis^59,60,61. This phenomenon has been extensively documented in various marine organisms, such as rainbow trout (Oncorhynchus mykiss)⁶², oyster (C. gigas)³⁰, abalone (Haliotis diversicolor⁶³ and Haliotis discus hannai⁶⁴). Additionally, the PI3K-AKT pathway can be activated by various membrane receptors, such as receptor tyrosine kinases (RTKs) and integrins (ITGs), which respond to thermal stress similarly to growth factor stimulation⁶⁵. A previous study revealed significant differences in the protein content and phosphorylation modifications of RTKs and ITGs, such as FGFR3 and ITGA9, between C. gigas and C. angulata during the thermal stress³⁰, further supporting the possibility of a high-temperature-RTK/ITG-PI3K-AKT-CASP3 pathway in oysters.

However, the CgAKT S464 residue (corresponding to HmAKT^S473), another crucial phosphorylation site, was not detected by western blotting due to its low sequence conservation. Despite this, the expression levels of mTOR, which is responsible for phosphorylating this site⁶⁶, and the phosphorylation levels at its critical site (CgmTOR^S2380 corresponding to HmmTOR^S2448; CgmTOR^S2412 corresponding to HmmTOR^S2481) showed a similar trends, with higher levels in C. angulata than in C. gigas after heat stress. Notably, the expression levels of phosphatase PP2A, which mediates the dephosphorylation of both CgAKT T291 and S464 sites, were consistent with our expectations. No significant changes in PP2A expression were observed during heat stress in C. gigas, in contrast, C. angulata showed a significant downregulation. This suggests higher kinase activity of CgAKT in C. angulata, leading to phosphorylation of CgCASP3 and resulting in lower CASP3 enzyme activity and apoptosis rates at high temperatures. Figure 6 provides a schematic of the molecular mechanisms underlying the divergent high-temperature-RTK/ITG-PI3K-AKT-CASP3 pathway between C. gigas and C. angulata during heat stress.

**Fig. 6: Schematic representation of PI3K-AKT-CASP3 axis of oysters in facing high temperature.**

Conclusion

In summary, this study identified a novel group of Bivalvia and Gastropoda Caspase-3/6/7 protein with a long IL that inhibits their activation. Within this region, a conserved phosphorylation modification at the Thr260 residue in oysters was found to be mediated by AKT kinase, which subsequently inhibited the heat-induced activation of novel Cgcaspase-3/6/7. Additionally, this pathway is regulated by the classic PI3K-AKT pathway. It shows divergent temperature-response patterns between two allopatric congeneric oyster species: the relatively cold-adapted C. gigas and the relatively warm-adapted C. angulata. This study characterized the function of these novel effector caspase members and their long IL in oysters, reporting for the first time that the PI3K-AKT pathway phosphorylates the conserved Thr260 in CgCASP3/6/7’s linker region to inhibit its heat-induced activation in non-model organisms. These findings reveal complex and unique apoptotic regulatory mechanisms in bivalves, providing new insights into the evolution and function of the crosstalk between the PI3K-AKT and caspase pathways.

Materials and methods

Animal material

Wild adult oyster of C. gigas and C. angulata were collected as broodstocks from Qingdao (35°44′ N) and Xiamen (24°33′ N), respectively, and transferred to Qingdao for conducting one-generation common garden experiment to alleviate environmental influences. The artificial breeding protocol including broodstock conditioning, fertilization, and larval cultures, all of which were conducted in hatchery with 22–26 °C and 31 ± 1‰ seawater. Briefly, thirty mature female eggs were mixed and divided into thirty beakers for each species. Sperm from each of the 30 mature males was crossed separately with the eggs in each beaker. Then, Juvenile F1 progeny (8 months old) of each species which were raised in the sea of Muping City (37◦39′N, Shandong province, China), were collected and brought to Qingdao for heat shock experiments. The samples of two oyster species were cleaned and acclimated in an aquarium with aerated and sand-filtered seawater for two weeks. Spirulina powder was added as food source, and the seawater was changed daily at the temperature of 18 ± 2 °C, which was the same as that of the Muping offshore area. After the acclimation period, oysters were exposed to seawater with a temperature of 37 °C (Sublethal Temperature) for 12 h⁶⁷. And the control group was placed in 18 ± 2 °C seawater. A total of 60 oyster gill tissues (15 C. gigas (HGi) and 15 C. angulata (HAn) under heat stress, 15 C. gigas (NGi) and 15 C. angulata (NAn) without treatment) were sampled and placed into liquid nitrogen, then stored in a − 80 °C refrigerator for subsequent experiments.

Phylogenetic analysis

The Caspase-3, Caspase-6 and Caspase-7 protein sequences of H. sapiens, M. musculus and D. rerio were retrieved from the National Center for Biotechnology Information (NCBI) database. The BLASTP analysis implemented in TBtools with an E-value threshold of 10⁻⁵ was used to identify all possible effector caspase proteins in other species’ amino acid sequence databases⁶⁸. Based on the results of domain identification by PROSITE, proteins containing both the P20 and P10 domains were manually selected and identified as potential effector caspase protein. The maximum likelihood phylogenetic tree was reconstructed using PhyloSuite 1.2.2 software⁶⁹, incorporating all inferred effector caspase amino acid sequences from both vertebrate and invertebrate species. Sequence alignment was performed using MAFFT software (https://www.ebi.ac.uk/Tools/msa/mafft/)⁷⁰, and poorly aligned sequences and gaps were removed using Gblocks 9.1b (http://www.phylogeny.fr/one_task.cgi?task_type=gblocks)⁷¹. The optimal model was determined using ModelFinder⁷², and the VT + G4 model was selected for multi-species tree reconstruction. IQ-TREE integrated in PhyloSuite was utilized for maximum likelihood tree construction with 1000 bootstrap replicates⁷³. HmCASP2 was designated as outgroup. The phylogenetic tree was visualized and beautified using the iTOL online instrument (http://itol.embl.de/). Collinearity analysis and the prediction of segmental duplications and tandem duplications of all effector caspase genes in C. gigas genome (GenBank accession no. GCA_011032805.1)⁷⁴ were performed and visualized by TBtools⁶⁸. See Supplementary Data 1 for details.

Gene expression analysis of all oyster effector caspase genes

The raw sequencing data of the oyster transcriptome in different tissues, developmental stages, and under various stress conditions were obtained from the Short Read Archive (SRA) database (SRP014559; SRP019967)^7,75. These data were aligned to the updated genome of C. gigas (GenBank accession number: GCA_011032805.1)⁷⁴ and quantified using the Salmon software (https://combine-lab.github.io/salmon/)⁷⁶. The heatmap was generated using the OmicShare tool (https://www.omicshare.com/tools).

In vitro DEVD-ase activity assay

The full-length CDS of Cg CASP3/6/7 like6 (referred to as CgCASP3 hereafter), as well as its middle region deletion mutant (K220-F339; CgCASP3-∆M)¹² and the Thr260 site-mutated variants, were amplified and inserted into the pET-32a plasmid for expression with the 6 X His-tags at the N- and C-ends (Supplementary Table 3). The 5 μg purified CgCASP3, CgCASP3-∆M, CgCASP3^T260A and CgCASP3^T260D were added into 40 μL Caspase activity buffer (50 mM Hepes (pH 7.4), 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA disodium salt, 0.10% Chaps, 10% glycerol, 10 μM ZnSO₄ and 50 μM PAC-1)⁷⁷ and incubated at 37 °C for 2 h. Then, 5 μL of 20 mM stock of Ac-YVAD-pNA, Ac-VDQQD-pNA, Ac-DEVD-pNA, Ac-LEVD-pNA, Ac-VEID-pNA, Ac-IETD-pNA and Ac-LEHD-pNA (Beyotime Biotechnology, China) were added into mixture as the substrate. Caspase inhibitor (Z-VAD-FMK) and Caspase-3/7 inhibitor (Ac-DEVD-CHO; Beyotime Biotechnology, China) were also added to assess its effect on CgCASP3 enzyme activity. Then, the absorbance of each well of the plate at 405 nm was read every 10 min using the Varioskan Flash multimode reader (Thermo Fisher Scientific, USA).

In vivo DEVD-ase activity assay

Recombinant mCherry-CgCasp3 or mCherry-CgCasp3-∆M and its Thr260 site-mutated variants, along with additional upstream plasmids such as Myc-CgAkt plasmid, were co-transfected into HEK293T cells using the Lipofectamine 3000 (Invitrogen, USA), which was cultured in high-glucose DMEM Medium (Biological Industries, Israel) and 10% fetal bovine serum (Biological Industries, Israel). After incubating with Apoptosis Inducer Kit (TNF-α + SM-164; Beyotime Biotechnology, China) for 8 h, the DEVD-ase from cells lysates of each group or gill tissues from C. gigas and C. angulata under heat stress were measured by the CASP3 kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions.

Cell counting kit-8 (CCK-8) assay

The HEK293T cells transfected as mentioned in the section “In Vivo Caspase-3 Activity Assay” were induced by Apoptosis Inducer Kit (TNF-α + SM-164) for 8 h. After 24 h, the cellular viability in each group was measured using the Enhanced Cell Counting Kit-8 (Beyotime Biotechnology, China) by the Varioskan Flash multimode reader at 450 nm.

Cell apoptosis assay

The HEK293T cells transfected as mentioned in the section “In Vivo DEVD-ase Activity Assay” were induced by Apoptosis Inducer Kit (TNF-α + SM-164) for 8 h. After 24 h, the cell apoptosis rate was measured using Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (Solarbio, China) according to the manufacturer’s instructions. And the apoptotic cells were detected using the FACSAria II flow cytometry (Becton Dickinson, USA). Data was analyzed using FlowJo (version 10.8.1).

Lactate dehydrogenase (LDH) detection

Cell transfection and apoptosis induction were same with the section “In Vivo DEVD-ase Activity Assay”. The release of LDH from each group was measured by CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Netherlands) using the Varioskan Flash multimode reader at 490 nm, according to the manufacturer’s instructions. The percent cytotoxicity was calculated using the following formula: percent cytotoxicity = 100 × (experimental sample - culture medium background)/(maximum LDH release - culture medium background).

TUNEL assay

Recombinant mCherry-CgCasp3 or mCherry-CgCasp3-∆M were transfected into HEK293T cells as above. The apoptosis induction was same with the section “In Vivo DEVD-ase Activity Assay”. The TUNEL assay was conducted using the One Step TUNEL Apoptosis Assay Kit (Beyotime Biotechnology, China), according to the manufacturer’s instructions. Briefly, the HEK293T cells from each group were fixed in 4% paraformaldehyde for 30 min at 37 °C, permeabilized in 0.1% Triton X-100 for 2 min and incubated with TUNEL assay reagents for 1 h at 37 °C. Images were captured using a confocal microscope LSM710 (Carl Zeiss, Germany) and analyzed by Image J software (Image J 1.53q; http://imagej.nih.gov/ij).

SDS-PAGE analysis of CgCASP3 activation

The 10 μg purified CgCASP3 and CgCASP3-∆M were added into 35 μL Caspase activity buffer and incubated at 37 °C for 2 h. Then, the reaction was stopped by the addition of 4X protein loading buffer (GenScript Biotech, China) and boiling for 10 min. A 40 μL volume of each sample was loaded on a 4–20% precast polyacrylamide gel. The gel was stained with Coomassie brilliant blue (Epizyme, China). The HEK293T cells transfection and apoptosis induction were same with the section “In Vivo DEVD-ase Activity Assay”. Additionally, the Akt/PKB inhibitor MK-2206 (HY-108232; MCE, USA) and activator SC79 (HY-18749; MCE, USA) in DMSO was prediluted with complete media and then added to the cells to reach a final concentration of 1, 3, 5 μM and 2, 5, 10 μM, respectively. Cells were subsequently placed in the cell culture incubator for 8 h. Then, the cells from each group were lysed by Cell lysis buffer for Western and IP (Beyotime Biotechnology, China) with protease and phosphatase inhibitors (Beyotime Biotechnology, China). The proteins were separated by 4–20% precast polyacrylamide gel and analyzed by western blotting.

Prediction of kinase

The iGPS1.0 software was utilized to predict potential kinases targeting the CgCASP3 Thr260 site. This prediction is based on the Short Linear Motif (SLM) theory, which focuses on short linear motifs surrounding phosphorylation sites (p-sites) and provides high specificity⁷⁸. The H. sapiens was chosen as the organism, and the threshold was set to “medium” with the “interaction” parameter configured as “Exp./String”.

Co-IP

Co-IP assays were conducted using the anti-mCherry magnetic beads Magnetic Beads (ABclonal, China). The full-length CDSs of CgCasp3 and CgAkt were amplified and inserted into pCMV-N-mCherry and pCMV-N-Myc plasmids for fusion with the tag, respectively (Supplementary Table 3). The cell transfection was same with above. After 36 h, the cells lysates were incubated with anti-mCherry magnetic beads and Mouse IgG magnetic beads overnight. Following washing step, the reaction products were loaded onto 4–20% precast polyacrylamide gel (GenScript Biotech, China), and then the signals were obtained by western blotting.

BiFC assay

The full-length CDSs of CgCasp3 and CgAkt were amplified and inserted into pBiFC-VN173 and pBiFC-VC155 plasmids, respectively (MiaoLing Plasmid Platform, China; Supplementary Table 3). Then, these plasmids were transfected into HeLa cells (Procell Life Science & Technology, China), which was cultured in RPMI Medium 1640 (Biological Industries, Israel) and 10% fetal bovine serum (Biological Industries, Israel). After 36 h, the fluorescence was imaged using a confocal microscope LSM710.

Yeast two-hybrid assay

The full-length CDSs of CgCasp3 and CgAkt were amplified and inserted into pGBK-T7 and pGAD-T7 (MiaoLing Plasmid Platform, China), respectively (Supplementary Table 3). Pairwise interactions were tested using GAL4 Yeast Two-Hybrid Media Kit (Coolaber, China). Briefly, each vector (bait and prey) was transformed into the Y2HGold yeast strain and initially plated on -Leu -Trp plates for the growth selection of transformants. After 2–3 days, the growing transformants were inoculated into (-Leu, -Trp) medium and continuously shaken overnight at 30 °C and 200 rpm. Subsequently, 10 μl of cell suspension (diluted in ddH₂O to OD 0.5 and 1.0) were plated onto selective plates (-Leu, -Trp), (-Leu, -Trp, -His3), and (-Leu, -Trp, -His3, -Ade2) and incubated for 2–3 days to assess the presence of interactions.

Subcellular localization

The full-length CDSs of CgCasp3 and CgAkt were amplified and inserted into pCMV-N-mCherry and pCMV-N-EGFP plasmids for fusion with the reporter gene, respectively (Supplementary Table 3; Beyotime Biotechnology, China). The cell culture, plasmid transfection and imaged for fluorescence as mentioned in the section “BiFC Assay”.

In vivo phosphorylation assay

Recombinant mCherry-CgCasp3 plasmid and its Thr260 site-mutated variants, along with additional upstream plasmids such as Myc-CgAkt plasmid, were co-transfected into HEK293T cells as above. After 36 h, the cells were lysed using a lysis buffer supplemented with protease and phosphatase inhibitors (Beyotime Biotechnology, China). The mCherry-CgCASP3 protein was subsequently purified using the anti-mCherry magnetic beads as mentioned in the section “Co-IP”. The phosphorylation levels of the CgCASP3 protein were then measured using western blotting with anti-phosphoserine/threonine antibody (ECM, PP2551; 1:1000 dilution).

In vitro kinase activity assay

The purified CgCASP3 and CgAKT and their site-mutated variants were used to test whether CgAKT could phosphorylate CgCASP3 Thr260 site, as well as to evaluate the influence of phosphorylation at the Ser464 and Thr291 sites of CgAKT on its ability to phosphorylate CgCASP3. The wild type and T260A mutant of CgCASP3 was used as substrate. The kinase activity assay was performed in 20 μl kinase buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, 20 mM ATP (CST, USA), 1 μg substrate protein and 1 μg kinase protein for 30 min at 30 °C. And the reactions were stopped with SDS loading buffer and measured by western blotting with anti-phosphoserine/threonine antibody.

Western blotting

The samples from oysters and cells were lysed with protease and phosphatase inhibitors. The supernatant proteins were collected by centrifugation and denatured at 100 °C for 10 minutes after adding 4X protein loading buffer. Subsequently, the proteins were transferred onto a 0.45 nm pore size polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the eBlot™ L1 wet transfer (GenScript, China). The membrane was then blocked and incubated with primary and secondary antibodies using the eZwest Lite Automated Western Device (GenScript, China). Afterwards, the membrane was incubated with the Omni-ECL™ Femto chemiluminescence kit (Epizyme, China) and captured using the Molecular Imager® Gel Doc™ XR system (Bio-Rad, USA). The following antibodies were used: mCherry-tag (ABclonal, AE094; 1:1000 dilution), Myc-tag (ZENBIO, 390003; 1:1000 dilution), beta Actin (ABclonal, AC026; 1:1000 dilution), Cleaved-PARP1 (ZENBIO, 380374; 1:1000 dilution), Flag-tag (ZENBIO, 390002; 1:1000 dilution), HA-tag (ZENBIO, 201113; 1:1000 dilution), His-tag (ZENBIO, 230001; 1:1000 dilution), AKT1 (ABclonal, A22533; 1:1000 dilution), Phospho-AKT1-T308 (ABclonal, AP1214; 1:1000 dilution), PDPK1 (ABclonal, A1665; 1:1000 dilution), Phospho-PDPK1-S241 (ABclonal, AP0426; 1:1000 dilution), PTEN (ABclonal, A19104; 1:1000 dilution) and PIK3CA (ABclonal, A16950; 1:1000 dilution), HRP-labeled Goat Anti-Mouse/Rabbit IgG(H + L) (Epizyme, LF101; 1:5000 dilution) and HRP-labeled Goat Anti-Mouse/Rabbit IgG(H + L) (Epizyme, LF102; 1:5000 dilution).

Statistics and reproducibility

All statistical analyses were performed using GraphPad Prism version 8.0.2 for Windows. The data were analyzed with the Mann-Whitney test and Brown-Forsythe and Welch ANOVA tests. Data are shown as the means ± SD, and the number of replicates (n) are denoted in the corresponding figure legends. Significant differences between groups were marked with “^*” for p < 0.05, “^**” for p < 0.01, “^***” for p < 0.001 and “^****” for p < 0.001. The schematic presentation was created using BioRender software (https://biorender.com).

In Figs. 2A, B, I, Fig. 3I, the purified CgCASP3 protein, the truncated mutant lacking IL, and the mutants with mutation at the T260 site were co-incubated with caspase substrates. Fluorescence values were recorded every ten minutes, with five biological replicates per group. In Figs. 2D–G, Fig, 3D–G, Fig. 4G–J and Fig. 5F–I, the HEK293T cells were transfected with pCMV-N-mCherry empty vector, mCherry-CgCasp3, mCherry-CgCasp3-∆M, mCherry-CgCasp3 T260 site mutants, Myc-CgAkt, and Myc-CgAkt T291 S464 sites mutants. Subsequently, all groups were co-incubated with TNF-α + SM-164 apoptotic inducers for 8 h. The cell-based transfection DEVD enzyme activity, cell viability, lactate dehydrogenase (LDH) release, and cell apoptosis rate were measured at 0 h, 2 h, 4 h, and 8 h time points, with 3 to 5 biological replicates per group. For all the experimental results, no data were excluded from the analysis.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Source data for all the figures in the manuscript can be found in Supplementary Data 2.

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Acknowledgements

We acknowledge the support from Oceanographic Data Center, IOCAS; Zhanhui Hou for its guidance and assistance in the use of flow cytometer and confocal microscopy. This research was funded by the National Key R&D Program of China (No. 2022YFD2400304), Key Research and Development Program of Shandong (2022LZGC015; ZFJH202309), and China Agriculture Research System of MOF and MARA (No. CARS-49).

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These authors contributed equally: Chaogang Wang, Mingyang Du.

Authors and Affiliations

Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture(CAS), Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
Chaogang Wang, Guofan Zhang & Li Li
Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, China
Chaogang Wang, Rihao Cong & Guofan Zhang
Shandong Province Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
Chaogang Wang, Mingyang Du, Zhuxiang Jiang, Rihao Cong, Wei Wang, Taiping Zhang, Jincheng Chen, Guofan Zhang & Li Li
National and Local Joint Engineering Laboratory of Ecological Mariculture, Qingdao, China
Chaogang Wang, Mingyang Du, Zhuxiang Jiang, Rihao Cong, Wei Wang, Taiping Zhang, Jincheng Chen, Guofan Zhang & Li Li
University of Chinese Academy of Sciences, Beijing, China
Mingyang Du, Zhuxiang Jiang, Taiping Zhang, Jincheng Chen & Li Li
Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang), Zhanjiang, China
Rihao Cong & Guofan Zhang
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao Marine Science and Technology Center, Qingdao, China
Wei Wang & Li Li
Shandong Technology Innovation Center of Oyster Seed Industry, Qingdao, China
Guofan Zhang & Li Li

Authors

Chaogang Wang
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Mingyang Du
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Zhuxiang Jiang
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Rihao Cong
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Wei Wang
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Taiping Zhang
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Jincheng Chen
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Guofan Zhang
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Li Li
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Contributions

L.L. and G.Z. conceived the study. C.W., M.D., Z. J., T. Z. and J. C. carried out the field and laboratory work, collected the oyster samples, participated in the data analysis, and drafted the manuscript. R.C. and W.W. contributed to cultural management. C.W., L.L. and G.Z. revised the manuscript. All authors approved the manuscript for publication.

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Correspondence to Li Li.

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Communications Biology thanks Susanne Vogeler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Toril Holien and David Favero.

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Wang, C., Du, M., Jiang, Z. et al. PI3K-AKT-mediated phosphorylation of Thr260 in CgCaspase-3/6/7 regulates heat-induced activation in oysters. Commun Biol 7, 1459 (2024). https://doi.org/10.1038/s42003-024-07184-4

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Received: 11 March 2024
Accepted: 31 October 2024
Published: 07 November 2024
DOI: https://doi.org/10.1038/s42003-024-07184-4

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