Molecular insight on the role of the phosphoinositide PIP₃ in regulating the protein kinases Akt, PDK1, and BTK Open Access

The phosphorylation of proteins and peptides at the Ser, Thr, and Tyr amino acids is essential in almost all eukaryotic cellular processes. This is catalyzed by the transfer of the terminal phosphate from ATP by the action of protein kinases, with dysregulation of kinase activity being causative of many human diseases. This has made them important targets in pharmaceutical development with >30 kinase inhibitors being clinically approved for a wide variety of diseases [1–3]. The human kinome is extensive with >530 different kinases, representing ~2% of the protein coding genes in the human genome [4]. These can be classified into Ser/Thr kinases, Tyr kinases, and pseudokinases [5]. Many mechanisms have evolved to tightly control the activity of kinases, making sure that they are turned on in the correct cellular context. This is crucial for the fidelity of kinases involved in signaling cascades, with many requiring multiple activating inputs for full activation. Kinases have an inherent specificity for their substrates encoded by their kinase domains [6,7], but multiple additional factors can control activity including scaffolding, cellular localization, protein–protein interactions, metabolites, and post-translational modifications [8,9]. One of the regulatory mechanisms that can control kinase activity and localization is the targeted binding to lipid phosphoinositides, frequently mediated by lipid-binding domains. This review will explore how lipid phosphoinositides can regulate different kinases' activity, with a specific focus on the molecular basis for how lipid-binding domains alter kinase activity by an analysis of previous biochemical/structural data in combination with recent advances in AlphaFold3-enabled protein modeling [10].

Defining the basis for how lipid-binding domains alter kinase activity requires discussion of the molecular mechanisms controlling kinase activation. The first protein kinase structure of the enzyme protein kinase A (PKA) was solved ~35 years ago using X-ray crystallography [11], followed by extensive studies using X-ray, NMR, and cryo-EM with now more than half of all human kinase domains being structurally characterized, in a variety of active and inactive states [1]. This has allowed for the identification of multiple conserved features found in almost all active conformations of protein kinases and the divergent mechanisms by which protein kinases can be inactivated. Kinases often follow the Anna Karenina principle: while their active conformations are structurally similar featuring a conserved catalytic architecture necessary for function, their inactive conformations are diverse. This principle suggests that activation requires a conserved set of structural features, whereas inactivation results from unique structural disruptions. As a result, inactive kinases adopt a broad array of conformations, reflecting the many different ways activation can be perturbed [12].

Due to space limitations, this review will only briefly survey these general mechanisms as they relate to lipid-regulated kinases, but readers are advised to consult previous reviews for a more in-depth analysis [1,8]. The kinase domain (KD) fold is a bi-lobal structure composed of an N-lobe and a C-lobe, with ATP binding at the cleft between these two lobes (Figure 1). In the active conformation, there are a set of hydrophobic interactions that occur in two spines that span the N- and C-lobes, with these being referred to as the regulatory and catalytic spines. Many aspects of kinase regulation are controlled by conformational changes between inactive states where the spines are disassembled. The region binding to ATP is often referred to as the hinge, with this being the connection point between the two lobes. The adenine ring of ATP forms part of the catalytic spine of hydrophobic interactions. Conserved features in protein kinases include the activation loop, containing residues that mediate binding to protein substrate. At the N-terminus of the activation loop, there is a strongly conserved DFG motif, with the aspartic acid playing an important role in co-ordinating the Mg²⁺ cofactor. The phenylalanine residue can exist in either a DFG-in or DFG-out conformation, with the DFG-in state being the active state. The change in orientation of this region of the activation loop plays a critical role in assembling the regulatory spine in the active conformation. Active kinase structures almost always contain a critical salt bridge from a glutamic acid in a conserved helix (αC helix) to a lysine in the β-sheet of the N-lobe. Together, these interactions are critical for orienting catalytic residues to maximize substrate catalysis. The activity of kinases can be modulated by modifying the conformation of the αC helix, mediated by post-translational modifications, protein–protein-binding partners, and intra-domain interactions.

This review focuses specifically on the structural basis of regulation of phosphoinositide-regulated kinases, specifically the AGC family kinases, Akt1/2 and 3-phosphoinositide-dependent kinase (PDK1), and the TEC family kinase, Bruton’s agammaglobulinemia tyrosine kinase (BTK). Specific regulatory features that control the activity of these kinases will be described in the following sections.

The AGC kinase family contains 63 serine/threonine kinases, including PDK1 and Akt/protein kinase B (PKB), which have highly conserved kinase domains [4]. PDK1 is a master regulator of cellular life [14]. When knocked out in mice, it results in early embryonic lethality, mainly driven by abnormalities in neuronal development [15]. PDK1 is described as a master regulator of kinase signaling, as it directly phosphorylates multiple AGC family kinases, including Akt/PKB [16], SGK, S6K [17], RSK [18], and PKC [19] kinase subfamilies. PDK1 is also able to phosphorylate and activate PKA [20]. However, PKA phosphorylation and activity are similar in both WT and PDK1 knockout embryonic stem cells [21], revealing that the ability to phosphorylate a substrate is not always directly linked to regulation. A unique regulatory feature of PDK1 is the presence of a PIFtide pocket within the N-lobe above the αC helix. This pocket is named for its ability to bind PDK1-interacting fragment (PIF) peptides, present in the hydrophobic motifs (HMs) of AGC substrate kinases [22,23]. This hydrophobic pocket plays a key allosteric role in regulating PDK1 activity, as it recruits downstream substrate kinases, and its occupancy by either substrates or small molecules leads to PDK1 activation [24].

Crystal structures of PDK1’s kinase domain in different activation states show that the conformation of the catalytic cleft, irrespective of whether it is non-phosphorylated [25] or phosphorylated in the activation loop (S241) [26], or in the presence or absence of peptides bound to the PIF pocket [27], is in its active conformation (Figure 2A–D). The most well-studied PIF pocket binder is known as PIFtide, which is derived from the HM of PRK2. While the binding of the PIFtide to PDK1’s PIF pocket does not change the architecture of the catalytic cleft, there are several hydrogen bonds established upon PIFtide binding with the periphery of the αC helix, as well as the β-sheet packing against the β-strand containing the catalytic lysine, consistent with observed increases in the specific activity of PDK1 when PIFtides are bound to the pocket [22].

While many of PDK1’s substrates are other AGC family kinases, different inputs are required for the phosphorylation of different substrates [14]. For instance, substrates such as SGK, S6K, and PKC require docking of their C-terminal PIFtide to PDK1’s PIF pocket dependent on HM phosphorylation [28]. In contrast, PDK1-mediated phosphorylation of the Akt activation loop requires PI(3,4,5)P₃ or PI(3,4)P₂ [29,30], with data suggesting that Akt’s HM does not associate with PDK1’s PIF pocket [31]. Interestingly, PDK1 and its substrate Akt share pleckstrin homology (PH) domains with similar specificity toward PI(3,4)P₂ and PI(3,4,5)P₃ phosphoinositides. C-terminal to its kinase domain is PDK1’s PH domain that interacts with phosphoinositides [32]. Critical to the regulation of PDK1 is dimerization mediated by the PH domain [33], which occurs upon binding to cellular membranes and is promoted by anionic lipids [34]. However, controversy exists around the role of these PH domains in regulating kinase activity [14,35,36], and how phosphoinositides alter this activity for both PDK1 and Akt. This raises possible questions about whether the PH domain functions as part of an autoinhibitory regulatory mechanism or if phosphoinositides primarily serve to bring PDK1 substrates into its proximity at the plasma membrane.

There is currently no high-resolution structural information for how PDK1 and its PH domain interact; however, there are multiple hydrogen deuterium exchange mass spectrometry (HDX-MS) studies of full-length PDK1 and the kinase domain alone [35,36]. We used AlphaFold3 to examine this HDX-MS data compared to these AI models (Figure 3A–C). Modeling was done in the presence of activation loop phosphorylation (pS241) as this is likely the best mimic of the biological conformation of PDK1. This was done in the context of multiple unique seeds, to add a level of randomness into the resulting models. In all cases, the PH domain was predicted to form a PH-KD interface at the C-lobe (Figure 3B and C). Intriguingly, the ensemble of predictions has different orientations of the PH domain, with ~30% projecting the phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) binding site toward the kinase domain, and 70% with it oriented away from the kinase domain. The interface with the C-lobe of the kinase domain is supported by recent HDX-MS analysis [35] (Figure 3D), with HDX-MS changes in the PH domain likely suggestive of a conformational ensemble (Figure 3E), as neither pose can fully explain the HDX-MS differences. However, we did not observe the previously described interface of the PH domain with the linker in any searchers [36], with this linker having very limited secondary structure as determined by HDX-MS in full-length PDK1 [35]. This ensemble of predicted PDK1 structures with the C-lobe of the kinase domain interacting with the PH domain agrees with a hypothesized interface and is validated by mutational analysis [37].

The role of the PH domain in regulating kinase activity, as well as the role of phosphoinositides in regulating phosphorylation, is controversial. Intriguingly, substrate phosphorylation occurs under different regulatory mechanisms, as disruption of PH-mediated dimerization prevents Akt phosphorylation but does not block S6K phosphorylation [36]. Further experiments fully investigating the role of the PH and kinase interface in controlling PDK1 activity will allow for the proving or disproving of the possible regulatory role of the PH domain in PDK1 signaling.

Akt (also referred to as protein kinase B, PKB) is a master regulator of cellular growth, metabolism, survival, and proliferation [38,39]. There are three isoforms of Akt (Akt1, Akt2, and Akt3), which are all activated downstream of phosphatidylinositol 3-kinase and remain in their inactive conformation in the absence of pro-growth signals. Akt activation occurs downstream of the second-most frequently mutated protein in human cancers (PIK3CA) and contains an activating oncogenic mutant (E17K) found primarily in breast and endometrial cancers. This makes it a promising therapeutic target, with the ATP-competitive inhibitor Capivasertib being FDA-approved for the treatment of hormone receptor-positive advanced breast cancer [40]. Like PDK1, Akt has a bi-lobal kinase domain housing similar catalytic machinery (Figure 4A and B). Unlike PDK1, Akt requires phosphorylation at two sites to adopt an active conformation, one in the activation loop (T308/309) by PDK1 [42,43] and the other in its C-terminal HM (S473/474) by mTORC2 [44] (Figure 4B and C). In the absence of these phosphorylation events, both the activation loop and HM are unstructured [41], along with a substantial region of the αC helix. Phosphorylation at the turn motif (T450/451) also occurs co-translationally to ensure correct folding of the bi-lobal kinase domain [45]. Unlike PDK1, Akt/PKB has no PIF pocket for their substrates to bind. Instead, its own phosphorylated HM binds the ‘PIF pocket’ (mediated by pS473/474), which, alongside activation loop phosphorylation at T308/309, drives the catalytic cleft to adopt an active conformation [13,41] through stabilization of the αC helix. To achieve full activation, Akt binds PIP₃ or phosphatidylinositol (3,4)-bisphosphate (PIP₂) at the plasma membrane through its PH domain [46]. However, whether PIP₃/PIP₂ binding is required once Akt is phosphorylated is still controversial. While kinase activity assays still show activation of fully phosphorylated Akt upon binding to PIP₃ membranes, the activity in the absence of membranes is still orders of magnitude higher than unphosphorylated Akt1 [47,48].

To understand the mechanism of how phosphoinositides activate Akt requires the understanding of how the PH domain interacts with the rest of the enzyme. N-terminal to the kinase domain is a PH domain (Figure 5A). While it is agreed upon that it forms an autoinhibitory interface with the kinase domain, the role it plays in inhibiting kinase activity following Akt phosphorylation is still contentious. The most controversial aspect being how Akt phosphorylates substrates that are distant from any membrane compartment that does not contain PI(3,4)P₂ or PIP₃, with there being two competing models: one where Akt is only active when phosphoinositides are present, and one where once phosphorylated, Akt can diffuse to its targets in the absence of lipids. Critical to testing this model is a rigorous evaluation of the molecular interactions that phosphoinositide lipids regulate, specifically the interface between the PH and kinase domains.

The first molecular insight into mapping the PH domain autoinhibitory interface was from the structure of Akt1 in complex with the allosteric inhibitor MK-2206 [51] that showed that the PIP-binding site in the PH domain was sequestered at the bi-lobal interface of Akt. However, it remained unknown if this allosteric compound locked the PH domain in a non-native inhibitory conformation. Years later, the X-ray crystallography structure of an engineered human Akt1 construct containing a shorter PH-kinase linker derived from Danio rerio bound to a stabilizing nanobody was solved, revealing an interface between the kinase domain’s C-lobe and PH domain with >500 Å of buried surface area [47] (Figure 5B). AlphaFold3 predictions showed a slightly different orientation of the PH domain compared with either of these models (Figure 5C–E). Across all three models, the PH domain engages primarily with the C-lobe of the kinase domain, forming an interface that occludes the PIP₃ binding pocket of the PH domain. While the general surface of the PH-kinase interaction is conserved across all three, the MK-2206-bound conformation exhibits the most deviation, where the PH domain is rotated and displaced relative to its position in the nanobody-stabilized and AlphaFold3-predicted structures. This unique orientation may reflect an inhibitor-induced conformational state that is either transient or less accessible under physiological conditions. Together, these structures suggest a model where the PH-KD autoinhibitory interface samples multiple conformations.

This is intriguing as it poses the question of whether the autoinhibitory PH-kinase interface can be maintained with and without regulatory phosphorylation, especially as the T308 site is located proximal to the interface with the PH domain. Work using protein semi-synthesis showed that phosphorylation of S473 relieves Akt autoinhibition through loss of an interaction with R144 in the PH-KD linker. Additionally, mutational analysis of R86 in the PH domain demonstrated decreased activity, suggesting it plays a role in establishing an autoinhibited Akt conformation [52]. Non-canonical HM phosphorylation events at S447 and T479 have also been proposed to relieve autoinhibition through a unique mechanism in the context of semisynthetic Akt [53]. However, HDX-MS studies argue that an autoinhibitory interface with the PH domain can be maintained in the T308/S473 phosphorylated state, as HDX-MS of a fully phosphorylated (T450, T308, S473) engineered Akt1 construct binding to 5% PIP₃ membranes showed changes consistent with breaking of the PH-kinase interface [47]. This included increases in exchange in the KD’s C-lobe and a decrease in deuterium exchange in the PH domain consistent with the removal of the PH domain caused by an interaction with PIP₃, with this occurring for both fully phosphorylated and monophosphorylated (T450) Akt1 (Figure 5,F and G). Interestingly, HDX-MS has shown a weakening of the PH-kinase autoinhibitory interface for both unphosphorylated and fully phosphorylated Akt1 binding to ATP-competitive inhibitors [54]. It is important to note that in both situations, the protein is either in the context of a truncated PH-kinase linker or protein semi-synthesis constructs that lack the critical T450 phosphosite. Therefore, fully resolving these conflicting biophysical results would require testing the activity and dynamics of both phosphorylated and non-phosphorylated states in as close to a biological mimic as possible using full-length Akt. It is possible that there are pools of Akt substrates that require phosphoinositides to both co-localize and maximally activate Akt activity, while other substrates can be phosphorylated by phosphorylated Akt once it is detached from membrane surfaces. This is consistent with recent work that showed differential phosphorylation of Akt substrates by distinct phospho-proteoforms, where different patterns of phosphorylation favored some Akt substrates over others [55].

BTK is a non-receptor tyrosine kinase in the TEC family kinase that plays a critical role in signal propagation downstream of the B-cell receptor, which is essential for B-cell development, differentiation, and signaling [56]. BTK is a therapeutic target in B-cell malignancies and autoimmune diseases, with multiple small-molecule inhibitors being FDA approved [57]. Inactivating mutations in BTK are causative of X‐linked agammaglobulinemia, leading to a significant decrease in mature B cells and frequent bacterial and viral infections. Structurally, BTK is regulated by its canonical Src module, consisting of a Src homology 2 (SH2), Src homology 3 (SH3), and kinase domain. The packing of the SH2 and SH3 domains against its SH3-kinase linker leads to the BTK kinase domain adopting an inactive conformation, with the αC helix rotated into an inactive state (Figure 6A - C) [58]. This inactive conformation of the Src module of BTK is similar to those of Src family kinases [60,61]. Disruption of the SH2 and SH3 interfaces leads to BTK’s kinase domain establishing its active conformation, where BTK can then become phosphorylated at Y551 in the activation loop by Src family kinases [62]. The active conformation of BTK bound to an ATP-competitive inhibitor is shown in Figure 6B - D[59], with the difference in conformation between inactive and active highlighted in Figure 6C and D.

N-terminal to the Src module is a PH domain and a Tec homology domain (defined as the PHTH module) (Figure 7A). However, the exact role of how these domains mediate BTK regulation is still not fully understood. The PH domain binds phosphatidylinositol (3,4,5)-trisphosphate (PIP₃) [64], with PIP₃ binding being essential for the membrane recruitment of BTK and its subsequent activation. PIP₃ binding results in dimerization of the PH domain and clustering of full-length BTK [65]. PH domain-mediated dimerization can also be promoted by soluble inositol hexakisphosphate (IP₆) [58] and is proposed to mediate activation by promoting trans-autophosphorylation of Y551. The role of the PHTH domains in regulating BTK in its inactive state remains unresolved. Crystal structures of full-length BTK show no clear density for the PHTH module, and low-resolution cryo-EM maps show multiple conformations of the PHTH relative to the Src module [63]. AlphaFold3 modeling using multiple seeds of full-length BTK (multiple seeds increases randomness) showed the PHTH domains sampling a large set of conformations, with limited high-confidence interactions occurring between these domains and the rest of the Src module (Figure 7C and D). HDX-MS analysis of full-length BTK versus the Src module of BTK revealed multiple regions with differences in exchange in multiple regions of the kinase domain, with these likely driven by HDX-MS sampling the conformational ensemble of PHTH conformations [66,67].

It has been proposed that the PH domain can bind to a partially active conformation of BTK, once the SH2 and SH3 domains are disengaged from the kinase domain. Structural insight into this has been provided by X-ray crystallography analysis of two constructs where the kinase domain is fused to the PHTH with different short linkers in the absence of the SH2+SH3 domains [58,63] (Figure 7B and C). In these structures, the PHTH domain was in an orientation incompatible with the presence of the SH2/SH3 domains. The use of different linkers revealed distinct orientations of the PHTH domain relative to the kinase domain, with both forming an interface with the N-lobe. This interface is likely dynamic and may serve to prevent full activation at low levels of PIP₃ by preventing BTK trans-autophosphorylation. Upon increased PIP₃ levels, the PHTH domain mediates dimerization, and BTK adopts a fully activated state (Figure 7E).

While the PH domains of Akt, PDK1, and BTK play important roles in regulating their activity, as discussed throughout the review, the occurrence of gain-of-function mutations at PH-KD interfaces is not uniformly shared. The most frequently observed oncogenic Akt mutation is E17K in Akt1, which is at the autoinhibitory interface between the kinase and PH domains. Its oncogenic mechanism is thought to involve enhanced phosphoinositide binding and increased membrane localization, without directly affecting kinase domain activity. However, this model remains under debate [52,68,69]. Equivalent mutations have not been reported in Akt2 or Akt3, which is likely due to their more restricted tissue distributions. Akt1 is ubiquitously expressed, whereas Akt2 is enriched in insulin-responsive tissues, and Akt3 is primarily found in brain, testis, and lung tissues. PDK1, although a central kinase in the PI3K/Akt pathway, has no reported oncogenic mutations. In BTK, disease-linked loss-of-function mutations are associated with X-linked agammaglobulinemia (XLA) [70,71]. Mutations in XLA patients contain missense, nonsense, deletions, and insertions, with some of these missense mutations clustering around the phosphoinositide-binding pocket of the PH domain, leading to reduced lipid binding and impaired BTK membrane localization [72], resulting in a loss of function. However, a distinct gain-of-function mutation, E41K, which is similar to that of Akt1’s E17K mutation, enhances phosphoinositide binding and drives constitutive membrane association [73,74]. It remains to be seen if this mutant has any influence on any PH domain autoinhibition of BTK.

Recent years have seen significant advances in our structural understanding of how lipid-binding PH domains regulate multiple different families of protein kinases. These studies have revealed both significant differences in the mechanisms of how phosphoinositide lipids can control kinase activity. Better tools to measure kinase signaling in different intracellular compartments, along with better tools for tracking and manipulating phosphoinositides, will allow for the full testing of different molecular models. Critical to the field is interpreting structural studies within the specific experimental caveats required to capture unique structural states that may exist in their biological context in a large conformational ensemble. There has been significant clinical progress being made in inhibiting PIP₃-regulated protein kinases, including multiple clinically approved inhibitors. It will be essential to fully work out the molecular basis of their activation in their native biological contexts, as this may be important to interpret the action of different ATP-competitive and allosteric inhibitors.

The authors declare no competing interests.

J.E.B. is supported by an operating grant from the Cancer Research Society (CRS- CRS-1052949). A.L.S. is supported by a Canadian Institute of Health Research (CIHR) CGS-D scholarship.

BCR

B-cell receptor

BTK

Bruton’s agammaglobulinemia tyrosine kinase

HDX-MS

hydrogen deuterium exchange mass spectrometry

HM

hydrophobic motif

PAE

Predicted aligned error

PDK1

3-phosphoinositide dependent kinase

PH

pleckstrin homology

PIF

PDK1 interacting fragment

PIP₂

phosphatidylinositol (3,4)-bisphosphate

PIP₃

phosphatidylinositol (3,4,5)-trisphosphate

PKA

protein kinase A

SH2

Src homology 2

SH3

Src homology 3

XLA

X-linked agammaglobulinemia

cryo-EM

cryogenic electron microscopy