Gregory J. Miller

The Expanding Roles of Gβγ Subunits in G Protein–Coupled Receptor Signaling and Drug Action

Gβγ subunits from heterotrimeric G proteins perform a vast array of functions in cells with respect to signaling, often independently as well as in concert with Gα subunits. However, the eponymous term “Gβγ” does not do justice to the fact that 5 Gβ and 12 Gγ isoforms have evolved in mammals to serve much broader roles beyond their canonical roles in cellular signaling. We explore the phylogenetic diversity of Gβγ subunits with a view toward understanding these expanded roles in different cellular organelles. We suggest that the particular content of distinct Gβγ subunits regulates cellular activity, and that the granularity of individual Gβ and Gγ action is only beginning to be understood. Given the therapeutic potential of targeting Gβγ action, this larger view serves as a prelude to more specific development of drugs aimed at individual isoforms.

Recognizing Phosphatidylinositol 3-Phosphate

Phosphatidylinositol 3-phosphate directs the endosomal localization of regulatory proteins by binding to FYVE and PX domains. New structures of these domains complexed with the phosphoinositide headgroup show how interactions with phosphate and hydroxyl groups differentiate this lipid from all others.

Recognition of accessory protein motifs by the gamma-adaptin ear domain of GGA3.

Adaptor proteins load transmembrane protein cargo into transport vesicles and serve as nexuses for the formation of large multiprotein complexes on the nascent vesicles. The γ-adaptin ear (GAE) domains of the AP-1 adaptor protein complex and the GGA adaptor proteins recruit accessory proteins to these multiprotein complexes by binding to a hydrophobic motif. We determined the structure of the GAE domain of human GGA3 in complex with a peptide based on the DFGPLV sequence of the accessory protein Rabaptin-5 and refined it at a resolution of 2.2 Å. The leucine and valine residues of the peptide are partly buried in two contiguous shallow, hydrophobic depressions. The anchoring phenylalanine is buried in a deep pocket formed by the aliphatic portions of two conserved arginine residues, along with an alanine and a proline, illustrating the unusual function of a cluster of basic residues in binding a hydrophobic motif.

Activin A Regulates Porcine Follicle-Stimulating Hormone β-Subunit Transcription via Cooperative Actions of SMADs and FOXL2

Activins stimulate FSH synthesis and secretion by pituitary gonadotrope cells. Activin A induction of porcine and murine FSHβ (Fshb) gene transcription in immortalized gonadotropes is dependent on homolog of Drosophila mothers against decapentaplegic (SMAD) proteins as well as the forkhead transcription factor L2 (FOXL2). Using both heterologous and homologous cell models, we demonstrate that FOXL2 functionally synergizes with SMAD3/4 to stimulate porcine Fshb promoter-reporter activity. We further show that endogenous FOXL2 and SMAD2/3 physically interact in homologous cells. We identify two composite cis-elements of adjacent FOXL2 and SMAD binding sites in the proximal porcine Fshb promoter that mediate activin A, FOXL2, and SMAD3 actions. FOXL2 can bind these elements independently of SMADs, whereas SMAD3/4 binding requires high-affinity FOXL2 binding. Conversely, FOXL2 alone is insufficient to regulate Fshb transcription and requires SMADs to induce promoter activity. Collectively, our data suggest a model in which activins stimulate formation and nuclear accumulation of SMAD3/4 complexes, which interact with FOXL2 bound to at least two proximal promoter elements. This association stabilizes SMAD3/4 binding to adjacent SMAD binding elements. SMAD-FOXL2 complexes then mediate activation of transcription through a currently unknown mechanism. Conservation of one of the two composite cis-elements suggests that this may form part of a general mechanism whereby activins regulate Fshb subunit transcription and FSH synthesis.

Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation

Background: The mechanism of substrate recognition for IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) is unresolved. Results: Binding and activity data reveal specific roles for each phosphate of IP5. Conclusion: The phosphate profile of IP5 is mechanistically critical to IPK1 activation. Significance: Identifying determinants of substrate specificity will aid in the design of selective inhibitors for IPK1. Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.

Inositol phosphate-induced stabilization of inositol 1,3,4,5,6-pentakisphosphate 2-kinase and its role in substrate specificity

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) on their inositol rings to yield an array of signaling molecules. IPKs must possess the ability to recognize their physiological substrates from among a pool of over 30 cellular IPs that differ in numbers and positions of phosphates. Crystal structures from IPK subfamilies have revealed structural determinants for IP discrimination, which vary considerably between IPKs. However, recent structures of inositol 1,3,4,5,6‐pentakisphosphate 2‐kinase (IPK1) did not reveal how IPK1 selectively recognizes its physiological substrate, IP5, while excluding others. Here, we report that limited proteolysis has revealed the presence of multiple conformational states in the IPK1 catalytic cycle, with notable protection from protease only in the presence of IP. Further, a 3.1‐Å crystal structure of IPK1 bound to ADP in the absence of IP revealed decreased order in residues 110–140 within the N‐lobe of the kinase compared with structures in which IP is bound. Using this solution and crystallographic data, we propose a model for recognition of IP substrate by IPK1 wherein phosphate groups at the 4‐, 5‐, and 6‐positions are recognized initially by the C‐lobe with subsequent interaction of the 1‐position phosphate by Arg130 that stabilizes this residue and the N‐lobe. This model explains how IPK1 can be highly specific for a single IP substrate by linking its interactions with substrate phosphate groups to the stabilization of the N‐ and C‐lobes and kinase activation.

PPIP5K1 interacts with the exocyst complex through a C-terminal intrinsically disordered domain and regulates cell motility.

Cellular signaling involves coordinated regulation of many events. Scaffolding proteins are crucial regulators of cellular signaling, because they are able to affect numerous events by coordinating specific interactions among multiple protein partners in the same pathway. Scaffolding proteins often contain intrinsically disordered regions (IDR) that facilitate the formation and function of distinct protein complexes. We show that PPIP5K1 contains an unusually long and evolutionarily conserved IDR. To investigate the biological role(s) of this domain, we identified interacting proteins using affinity purification coupled with mass spectrometry. Here, we report that PPIP5K1 is associated with a network of proteins that regulate vesicle-mediated transport. We further identified exocyst complex component 1 as a direct interactor with the IDR of PPIP5K1. Additionally, we report that knockdown of PPIP5K1 decreases motility of HeLa cells in a wound-healing assay. These results suggest that PPIP5K1 might play an important role in regulating function of exocyst complex in establishing cellular polarity and directional migration of cells.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Background: The phosphate profile of the inositol phosphate (IP) substrate defines inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) specificity. Results: IP substrate specificity is linked to IPK1 stability. Conclusion: IPK1 employs a mechanism of IP-induced stabilization to selectively recognize IP5. Significance: The IP recognition mechanism of IPK1 can be exploited to selectively target IPK1 for the study of higher IPs. Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

PPIP5K1 Suppresses Etoposide-triggered Apoptosis

Inositol hexakisphosphate kinase 2 (IP6K2) potentiates pro-apoptotic signalling and increases the sensitivity of mammalian cells to cytotoxic agents. Diphosphoinositol pentakisphosphate kinase (PPIP5K) generates inositol pyrophosphates (InsPPs) that are structurally distinct from those produced by IP6K2 and their possible roles in affecting cell viability remain unclear. In the present study, we tested the impact of PPIP5K1 on cellular sensitivity to various genotoxic agents to determine if PPIP5K1 and IP6K2 contribute similarly to apoptosis. We observed that PPIP5K1 overexpression decreased sensitivity of cells toward several cytotoxic agents, including etoposide, cisplatin, and sulindac. We further tested the impact of PPIP5K1 overexpression on an array of apoptosis markers and observed that PPIP5K1 decreased p53 phosphorylation on key residues, including Ser-15, -46, and -392. Overexpression of a kinase-impaired PPIP5K1 mutant failed to protect cells from apoptosis, indicating this protection is a consequence PPIP5K1 catalytic activity, in contrast with the sensitivity conferred by IP6K2, which is dependent on both catalytic and non-catalytic functions. These observations reveal distinct roles for PPIP5K1 and IP6K2 and the InsPPs they produce in controlling cell death.

Mass spectrometry analysis of PPIP5K1 interactions and data on cell motility of PPIP5K1-deficient cells

Inositol pyrophosphates are cellular signals that are created by the actions of inositol kinases and are degraded by highly active inositol phosphatases. The potent actions of these phosphatases suggest these signals must be created near their sites of action. To identify sites where the inositol kinase, PPIP5K1 acts, we performed affinity purification of PPIP5K1 from HEK293 cells and analyzed these samples using mass spectrometry to identify the proteins pesent (10.1016/j.cellsig.2016.02.002) [1]. We further decreased PPIP5K1 levels in HeLa cells and treated these with PPIP5K1 siRNA. We then monitored the motility of these cells in Scratch assays.