Type one gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling.

Type one gamma phosphatidylinositol phosphate kinase i5 suppresses YAP1 signaling.

Dysregulation of the Hippo/Yes-associated protein signaling pathway has been associated with several diseases, including cancer, neurological disorders, and cardiovascular conditions. However, the precise molecular mechanisms governing Hippo/YAP signaling are not fully understood, and additional regulators of this pathway need to be identified. Our research has identified type one gamma phosphatidylinositol phosphate kinase i5 as a regulator of Hippo/YAP signaling. PIPKIyi5 is a kinase responsible for synthesizing phosphatidylinositol-4,5-bisphosphate. By directly interacting with YAP1, PIPKIyi5 prevents the nuclear translocation of YAP1, thereby suppressing YAP1-mediated gene transcription. Thus, PIPKIyi5 functions as a suppressor of YAP1-mediated signaling. The kinase activity of PIPKIyi5, which generates phosphatidylinositol-4,5-bisphosphate, is essential for controlling YAP1 function. Phosphatidylinositol-4,5-bisphosphate promotes the interaction of YAP1 with the fourteen-three-three protein, which retains YAP1 in the cytosol. Given the role of YAP1 signaling in cancer cell stemness, depletion of PIPKIyi5 enhances YAP1 signaling and promotes tumor-sphere formation in head and neck squamous cell carcinoma. These findings highlight a phosphatidylinositol-4,5-bisphosphate-modulated signaling nexus that exerts specific control over Hippo/YAP signaling and its biological functions.

The Yes-associated protein one is a transcriptional coactivator and a key component of the Hippo/YAP pathway. This pathway is essential for maintaining normal tissue development by regulating cell contact-mediated inhibitory signaling. Upon activation, YAP1 translocates from the cytosol to the nucleus, where it associates with transcriptional enhanced associate domain family transcription factors to mediate the expression of target genes. Through this mechanism, YAP1 signaling governs processes, such as cell proliferation, differentiation, and organ regeneration. The activation of YAP1 must be tightly controlled, as its dysregulation is implicated in various diseases, including cancer, cardiovascular diseases, neurological disorders, and immune dysfunction. Notably, YAP1 is hyperactivated in many cancers, where it sustains stem cell properties and enhances cancer stem cell traits, contributing to therapeutic resistance, recurrence, and metastasis. As a result, YAP1-targeting strategies have emerged as a focus for anti-cancer therapies. Current approaches primarily aim to disrupt the YAP1-transcriptional enhanced associate domain interaction. However, the clinical success of these inhibitors has been limited. This highlights an unmet need for a deeper understanding of the regulatory mechanisms governing YAP1 activity and the identification of novel regulators as potential drug targets.

Phosphatidylinositol-4,5-bisphosphate is a vital component of membrane phospholipids. It serves not only as a precursor for producing key signaling molecules, such as phosphatidylinositol-3,4,5-trisphosphate, inositol-one, four, five-trisphosphate, and diacylglycerol but also functions as a critical messenger itself. By interacting with effector proteins and modulating their biological activities, phosphatidylinositol-4,5-bisphosphate regulates a wide range of cellular processes, including vesicular trafficking, cytoskeletal rearrangement, ion channel regulation, cell growth, and protein metabolism. Recent evidence suggests that YAP1 is a phosphatidylinositol-4,5-bisphosphate effector protein. Phosphatidylinositol 4-phosphate 5-kinase type one a, a kinase responsible for generating phosphatidylinositol-4,5-bisphosphate, plays a critical role in this context. In the nucleus, phosphatidylinositol 4-phosphate 5-kinase type one a interacts with YAP1, and the phosphatidylinositol-4,5-bisphosphate it produces facilitates the interaction of YAP1 with transcriptional enhanced associate domain transcription factors, thereby enhancing YAP1-transcriptional enhanced associate domain-mediated gene transcription. This indicates that nuclear phosphatidylinositol-4,5-bisphosphate can promote the activation of YAP1 signaling.

Phosphatidylinositol 4-phosphate 5-kinase type one a belongs to the type one phosphoinositide phosphate kinase family, which catalyzes the phosphorylation of phosphatidylinositol 4-phosphate to generate phosphatidylinositol-4,5-bisphosphate. In addition to phosphatidylinositol 4-phosphate 5-kinase type one a, the phosphatidylinositol 4-phosphate 5-kinase family includes two other isoforms, phosphatidylinositol 4-phosphate 5-kinase beta and phosphatidylinositol 4-phosphate 5-kinase gamma. These isoforms exhibit distinct subcellular localizations. Interestingly, phosphatidylinositol 4-phosphate 5-kinase type one a- or phosphatidylinositol 4-phosphate 5-kinase beta-knockout mice are viable and can survive into adulthood. In contrast, pan-knockout mice for phosphatidylinositol 4-phosphate 5-kinase gamma experience perinatal lethality, suggesting that phosphatidylinositol 4-phosphate 5-kinase gamma has unique biological functions that cannot be compensated by other phosphatidylinositol 4-phosphate 5-kinase isoforms, despite all isoforms contributing to phosphatidylinositol-4,5-bisphosphate production. The phosphatidylinositol 4-phosphate 5-kinase gamma gene undergoes alternative splicing, resulting in multiple splice variants. At least six variants named phosphatidylinositol 4-phosphate 5-kinase gamma one through phosphatidylinositol 4-phosphate 5-kinase gamma six are expressed in humans. These variants share the same N-terminal region and kinase domain but differ in their far C-terminal extensions. The distinct C-terminal regions allow these phosphatidylinositol 4-phosphate 5-kinase gamma variants to interact with specific effector proteins, enabling their recruitment to particular subcellular locations where they perform diverse biological functions.

but differ in their far C-terminal extensions. The distinct C- terminal regions allow these PIPKIY variants to interact with specific effector proteins, enabling their recruitment to particular subcellular locations where they perform diverse biological functions (30-34).

Intriguingly, our current research demonstrates that phosphatidylinositol 4-phosphate 5-kinase gamma five, a splice variant of phosphatidyinol 4-phosphate 5-kinase gamma, plays a distinct role in modulating YAP1 signaling compared with phosphatidyinol 4-phosphate 5-kinase alpha. While phosphatidyinol 4-phosphate 5-kinase alpha promotes YAP1 signaling in the nucleus, phosphatidyinol 4-phosphate 5-kinase gamma five modulates YAP1 in the cytosol. The production of phosphatidyinol-4,5-bisphosphate by phosphatidyinol 4-phosphate 5-kinase gamma five enhances the interaction between YAP1 and fourteen-three-three, effectively sequestering YAP1 in the cytosol. Consequently, phosphatidyinol 4-phosphate 5-kinase gamma five inhibits YAP1 nuclear translocation and suppresses YAP1-mediated gene transcription. These findings highlight that different phosphatidyinol-4,5-bisphosphate-producing kinases can modulate YAP1 signaling in a subcellular location-dependent manner.

Results

YAP1 specifically interacts with phosphatidyinol 4-phosphate 5-kinase gamma five.

During the investigation of potential PIPKIYi5 effector proteins, we identified an interaction between YAP1 and PIPKIYi5. As shown in Figure one A, FLAG-tagged YAP1 was coexpressed with HA-tagged PIPKIYi5 in human embryonic kidney two nine three cells, and a coimmunoprecipitation assay demonstrated that FLAG-YAP1 could be coimmunoprecipitated with HA-PIPKIYi5. This result indicates that PIPKIYi5 interacts with YAP1. To further validate the PIPKIYi5-YAP1 interaction, endogenous YAP1 was immunoprecipitated from CAL twenty seven cell lysates, and endogenous PIPKIYi5 was detected within the YAP1 complex via Western blot analysis (Figure one B). In addition, to determine whether PIPKIYi5 directly binds to YAP1, purified FLAG-YAP1 and HA-PIPKIYi5 recombinant proteins were used in the in vitro binding assay. As shown in Figure one C, purified HA-PIPKIYi5 was pulled down by purified FLAG-YAP1, confirming a direct interaction between PIPKIYi5 and YAP1.

To determine the specificity of the PIPKIYi5-YAP1 interaction, we compared the ability of other PIPKIY splice variants, such as PIPKIYil and PIPKIYi2, to interact with YAP1. As illustrated in Figure one D, these PIPKIY splice variants differ in the number of amino acids: PIPKIYil has six hundred forty amino acids, PIPKIYi2 has six hundred sixty-eight amino acids, and PIPKIYi5 has seven hundred seven amino acids. Notably, all three variants share an identical sequence for the first six hundred forty amino acids, whereas PIPKIYi2 and PIPKIYi5 have distinct C-terminal extensions built upon the core structure of PIPKIYil (Figure one D). Our results demonstrate that PIPKIYil does not bind to YAP1, and the interaction of PIPKIYi2 with YAP1 is significantly weaker compared with PIPKIYi5 (Figures one E and F). These findings confirm the specificity of the PIPKIYi5-YAP1 interaction and indicate that the unique C-terminal region of PIPKIYi5 is critical for its ability to bind YAP1. In addition, a kinase-dead mutant of PIPKIYi5 (D three one six A; referred to as PIPKIYi5KD) showed a marked deficiency in binding YAP1 (Figures one E and F). YAP1 is a PI four five P two effector protein capable of binding PI four five P two, which facilitates its interaction with the effectors such as TEAD family transcription factors. It is possible that the kinase activity of PIPKIYi5, which is responsible for generating PI four five P two, could regulate the PIPKIYi5 interaction with YAP1. To test this, a solid-phase in vitro binding assay was performed using purified recombinant FLAG-YAP1 and HA-PIPKIYi5 proteins to examine the effects of PI four five P two on YAP1-PIPKIYi5 interaction. As shown in Figures one G and H, the addition of PI four five P two significantly enhanced the interaction between YAP1 and PIPKIYi5. As a control, another phosphoinositide, phosphatidylinositol three five bisphosphate (PI three five P two), was tested and found unable to enhance the interaction between YAP1 and PIPKIYi5, demonstrating the specificity of PI four five P two in modulating the YAP1-PIPKIYi5 interaction (Figures one G and H).