PDK1-interacting Fragment Research Articles

Abstract 392: TAT-PHLPP9c and TAT-PIF Administration During CPR Synergistically Improves Cardiac Arrest Survival

Introduction: We have demonstrated that a novel fast-acting peptide TAT-PHLPP9c improves survival in both mouse and swine models of cardiac arrest. The protective effect is mediately by activation of Akt via inhibiting phosphatase PHLPP. We have designed another peptide TAT-PIF to activate Akt by promoting the active conformation of PDK1 via binding to PDK1 Interacting Fragment (PIF). Hypothesis: We hypothesize that TAT-PHLPP9c and TAT-PIF synergistically enhance Akt activation and improve cardiac arrest survival when administered intravenously during CPR. Methods: Cardiomyocytes were treated with TAT-PHLPP9c (10uM), or TAT-PIF (10uM) or in combination. Akt phosphorylation (p-Akt) at both Thr308 and Ser473 sites were assessed by Western blot. In C57BL/6 retired female mice (n=24), a 12-min asystolic cardiac arrest was induced with KCl followed by CPR and blinded randomized administration of the peptides or saline control. Four experimental groups were included: 1) saline, 2) TAT-PHLPP9c (7.5 mg/kg), 3) TAT-PIF (7.5 mg/kg), 4) TAT-PHLPP9c and TAT-PIF. Survival was evaluated at 4 hour post-ROSC. Results: In cardiomyocytes, compared to saline, both TAT-PHLPP9c and TAT-PIF independently increased AktSer473 phosphorylation, and the combined treatment of both peptides synergistically enhanced p-AktSer473. For AktThr308 site, TAT-PIF increased its phosphorylation while TAT-PHLPP9c induced a slight increase of its phosphorylation. Enhanced phosphorylation was also seen with the combined treatment. In mice, compared to saline control, both peptides improved 4-h survival when given alone, but the combined treatment improved 4-h survival synergistically over 30%. Conclusions: The cocktail of TAT-PHLPP9c and TAT-PIF may be a novel treatment regimen for improving outcome following cardiac arrest.

Single-molecule studies reveal regulatory interactions between master kinases PDK1, AKT1, and PKC

Leukocyte migration is controlled by a leading-edge chemosensory pathway that generates the regulatory lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), a growth signal, thereby driving leading-edge expansion up attractant gradients toward sites of infection, inflammation, or tissue damage. PIP3 also serves as an important growth signal in growing cells and oncogenesis. The kinases PDK1, AKT1 or PKB, and PKCα are key components of a plasma-membrane-based PIP3 and Ca2+ signaling circuit that regulates these processes. PDK1 and AKT1 are recruited to the membrane by PIP3, whereas PKCα is recruited to the membrane by Ca2+. All three of these master kinases phosphoregulate an array of protein targets. For example, PDK1 activates AKT1, PKCα, and other AGC kinases by phosphorylation at key sites. PDK1 is believed to form PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by a PDK1-interacting fragment (PIF) interaction between the PDK1 PIF pocket and the PIF motif of the AGC binding partner. Here, we present the first, to our knowledge, single-molecule studies of full-length PDK1 and AKT1 on target membrane surfaces, as well as their interaction with full-length PKCα. These studies directly detect membrane-bound PDK1-AKT1 and PDK1-PKCα heterodimers stabilized by PIF interactions formed at physiological ligand concentrations. PKCα exhibits eightfold higher PDK1 affinity than AKT1 and can competitively displace AKT1 from PDK1-AKT1 heterodimers. Ensemble activity measurements under matched conditions reveal that PDK1 activates AKT1 via a cis mechanism by phosphorylating an AKT1 molecule in the same PDK1-AKT1 heterodimer, whereas PKCα acts as a competitive inhibitor of this phosphoactivation reaction by displacing AKT1 from PDK1. Overall, the findings provide insights into the binding and regulatory interactions of the three master kinases on their target membrane and suggest that a recently described tumor suppressor activity of PKC isoforms may arise from its ability to downregulate PDK1-AKT1 phosphoactivation in the PIP3-PDK1-AKT1-mTOR pathway linked to cell growth and oncogenesis.

Single Molecule Studies of a Membrane-Bound, Master Kinase Circuit... Detecting Interactions of Two Master Kinase Pairs on the Membrane, PDK1-AKT1 and PDK1-PKC Alpha

In chemotaxing ameboid cells, a leading edge signaling network formed on the cytoplasmic leaflet of the plasma membrane directs actin and membrane remodeling to propel the cell up an attractant gradient. AKT1 (PKB) and PKCα kinases are central to the regulation of this chemotactic and other signaling pathways. To achieve full catalytic competence, PKCα and AKT1 are phosphorylated at key, C-terminal residues by PDK1, a master regulator of several AGC-family kinase members that control cell growth, proliferation, survival regulate metabolism. Interactions of AKT1, PDK1 and PKCα with the membrane are controlled by regulated generation of the signaling phosphoinositide lipid PIP3 (AKT1 and PDK1) and the influx of intracellular calcium (PKCα). Both AKT1 and PKCα possess a C-terminal PIF (PDK1 Interacting Fragment) motif thought to interact with a “PIF pocket” at the PDK1 catalytic domain. Productive collisions between membrane-bound AKT1:PDK1 and PKCα:PDK1 likely accelerate formation of the PIF:PIP pocket complex such that activating phosphorylation events by PDK1 at PKCα and AKT1 more efficient. Despite convincing work describing complexes between these regulatory kinases in solution, there has not been any evidence of AKT1:PDK1 or PKCα:PDK1 complex formation on a lipid membrane. The present work investigates the complexes between AKT1, PKCα and PDK1 using single molecule TIRF microscopy. The poster will present the findings of our work that will test the hypothesis that AKT1 and PKCα are each able to form a stable complex with PDK1, and the complex can be dissociated by adding a PIF peptide mimic. Moreover, by monitoring complex formation via increased protein:membrane friction and diffusional slowing, the affinity of each interaction can be derived. These findings are expected to yield new insight into the PDK1-dependent maturation of AKT1 and PKCα and may also assist with the development and testing of new therapeutics directed towards the kinases and kinase complexes.

Molecular Mechanism of Regulation of the Atypical Protein Kinase C by N-terminal Domains and an Allosteric Small Compound

Protein kinases play important regulatory roles in cells and organisms. Therefore, they are subject to specific and tight mechanisms of regulation that ultimately converge on the catalytic domain and allow the kinases to be activated or inhibited only upon the appropriate stimuli. AGC protein kinases have a pocket in the catalytic domain, the PDK1-interacting fragment (PIF)-pocket, which is a key mediator of theactivation. We show here that helix αC within the PIF-pocket of atypical protein kinase C (aPKC) is the target of the interaction with its inhibitory N-terminal domains. We also provide structural evidence that the small compound PS315 is an allosteric inhibitor that binds to the PIF-pocket of aPKC. PS315 exploits the physiological dynamics of helix αC for its binding and allosteric inhibition. The results will support research on allosteric mechanisms and selective drug development efforts against PKC isoforms.

The activation loop of PKA catalytic isoforms is differentially phosphorylated by Pkh protein kinases in Saccharomyces cerevisiae

PDK1 (phosphoinositide-dependent protein kinase 1) phosphorylates and activates PKA (cAMP-dependent protein kinase) in vitro. Docking of the HM (hydrophobic motif) in the C-terminal tail of the PKA catalytic subunits on to the PIF (PDK1-interacting fragment) pocket of PDK1 is a critical step in this activation process. However, PDK1 regulation of PKA in vivo remains controversial. Saccharomyces cerevisiae contains three PKA catalytic subunits, TPK1, TPK2 and TPK3. We demonstrate that Pkh [PKB (protein kinase B)-activating kinase homologue] protein kinases phosphorylate the activation loop of each Tpk in vivo with various efficiencies. Pkh inactivation reduces the interaction of each catalytic subunit with the regulatory subunit Bcy1 without affecting the specific kinase activity of PKA. Comparative analysis of the in vitro interaction and phosphorylation of Tpks by Pkh1 shows that Tpk1 and Tpk2 interact with Pkh1 through an HM-PIF pocket interaction. Unlike Tpk1, mutagenesis of the activation loop site in Tpk2 does not abolish in vitro phosphorylation, suggesting that Tpk2 contains other, as yet uncharacterized, Pkh1 target sites. Tpk3 is poorly phosphorylated on its activation loop site, and this is due to the weak interaction of Tpk3 with Pkh1 because of the atypical HM found in Tpk3. In conclusion, the results of the present study show that Pkh protein kinases contribute to the divergent regulation of the Tpk catalytic subunits.

Yeast 3-Phosphoinositide-dependent Protein Kinase-1 (PDK1) Orthologs Pkh1–3 Differentially Regulate Phosphorylation of Protein Kinase A (PKA) and the Protein Kinase B (PKB)/S6K Ortholog Sch9

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.

Turning a protein kinase on or off from a single allosteric site via disulfide trapping

There is significant interest in identifying and characterizing allosteric sites in enzymes such as protein kinases both for understanding allosteric mechanisms as well as for drug discovery. Here, we apply a site-directed technology, disulfide trapping, to interrogate structurally and functionally how an allosteric site on the Ser/Thr kinase, 3-phosphoinositide-dependent kinase 1 (PDK1)--the PDK1-interacting-fragment (PIF) pocket--is engaged by an activating peptide motif on downstream substrate kinases (PIFtides) and by small molecule fragments. By monitoring pairwise disulfide conjugation between PIFtide and PDK1 cysteine mutants, we defined the PIFtide binding orientation in the PIF pocket of PDK1 and assessed subtle relationships between PIFtide positioning and kinase activation. We also discovered a variety of small molecule fragment disulfides (< 300 Da) that could either activate or inhibit PDK1 by conjugation to the PIF pocket, thus displaying greater functional diversity than is displayed by PIFtides conjugated to the same sites. Biochemical data and three crystal structures provided insight into the mechanism of action of the best fragment activators and inhibitors. These studies show that disulfide trapping is useful for characterizing allosteric sites on kinases and that a single allosteric site on a protein kinase can be exploited for both activation and inhibition by small molecules.

Discovery of PDK1 Kinase Inhibitors with a Novel Mechanism of Action by Ultrahigh Throughput Screening

The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, K(m) values were determined as 5.6 mum for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1x and 10x K(m) concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1.

Identification of Allosteric PIF‐Pocket Ligands for PDK1 using NMR‐Based Fragment Screening and 1H‐15N TROSY Experiments

Aberrant activation of the phosphoinositide 3-kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3-phosphoinositide-dependent kinase-1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N-terminal lobe of the 3-phosphoinositide-dependent kinase-1 catalytic domain contains a docking site which recognizes the non-catalytic C-terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so-called PDK1 Interacting Fragment pocket allows interaction with 3-phosphoinositide-dependent kinase-1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3-phosphoinositide-dependent kinase-1 domain construct against a library of chemically diverse fragments in order to identify small, ligand-efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP-site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand-induced changes in 1H-15N TROSY spectra acquired using uniformly 15N-enriched PDK1 provided evidence to distinguish ATP-site from PDK1 Interacting Fragment-site binding. Caliper assay data and 19F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.

MUC1 is a target of hypoxia-inducible factor transcription factor in renal clear carcinomatous cells

Background: Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown.Results: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBα, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBα to one that phosphorylates both Thr308 and Ser473 of PKBα in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P3 to a form that is activated threefold by PtdIns(3,4,5)P3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBα in a PtdIns(3,4,5)P3-dependent manner and that is immunoprecipitated with PDK1 antibodies.Conclusions: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.

Structural and Functional Insights into the Regulation of Arabidopsis AGC VIIIa Kinases

The AGCVIIIa kinases of Arabidopsis are members of the eukaryotic PKA, PKG, and PKC group of regulatory kinases. One AGCVIIIa kinase, PINOID (PID), plays a fundamental role in the asymmetrical localization of membrane proteins during polar auxin transport. The remaining 16 AGCVIIIa genes have not been associated with single mutant phenotypes, suggesting that the corresponding kinases function redundantly. Consistent with this idea, we find that the genes encoding the Arabidopsis AGCVIIIa kinases have spatially distinct, but overlapping, expression domains. Here we show that the majority of Arabidopsis AGCVIIIa kinases are substrates for the 3-phosphoinositide-dependent kinase 1 (PDK1) and that trans-phosphorylation by PDK1 correlates with activation of substrate AGCVIIIa kinases. Mutational analysis of two conserved regulatory domains was used to demonstrate that sequences located outside of the C-terminal PDK1 interaction (PIF) domain and the activation loop are required for functional interactions between PDK1 and its substrates. A subset of GFP-tagged AGCVIIIa kinases expressed in Saccharomyces cerevisiae and tobacco BY-2 cells were preferentially localized to the cytoplasm (AGC1-7), nucleus (WAG1 and KIPK), and the cell periphery (PID). We present evidence that PID insertion domain sequences are sufficient to direct the observed peripheral localization. We find that PID specifically but non-selectively binds to phosphoinositides and phosphatidic acid, suggesting that PID might directly interact with the plasma membrane through protein-lipid interactions. The initial characterization of the AGCVIIIa kinases presented here provides a framework for elucidating the physiological roles of these kinases in planta.

Evaluation of Approaches to Generation of Tissue-specific Knock-in Mice

We explored three approaches to create tissue-specific knock-in mice by generating knock-in mice in which a substrate-docking site of the PDK1 protein kinase was ablated in Cre-expressing tissues in a way that prevented activation of one of its substrates, p70 ribosomal S6 kinase (S6K), but not another (protein kinase B (PKB)). Employing two of the approaches, termed the "heterozygous" and "minigene" methods, we generated mice in which Cre-expressing skeletal and cardiac muscle produced the mutant rather than wild type PDK1. Consistent with this, injection of these mice with insulin only induced activation of PKB but not S6K in muscle tissues. We have also demonstrated that insulin-stimulated glucose uptake proceeds normally in knock-in mice, consistent with the notion that PKB mediates this process. In contrast to conditional knock-out of PDK1 in muscle, the knock-in mice did not develop dilated cardiomyopathy, suggesting that PKB plays a key role in protecting mice from heart failure. The third knock-in strategy that was evaluated, termed the "inversion" method, did not proceed with high efficiency. We discuss the merits and disadvantages of each of the conditional knock-in approaches, along with the applications for which they may be most suited, and suggest how they could be further refined.

Steady-state Kinetic Mechanism of PDK1

PDK1 catalyzes phosphorylation of Thr in the conserved activation loop region of a number of its downstream AGC kinase family members. In addition to the consensus sequence at the site of phosphorylation, a number of PDK1 substrates contain a PIF sequence (PDK1-interacting fragment), which binds and activates the kinase domain of PDK1 (PDK1(deltaPH)). To gain further insight to PIF-dependent catalysis, steady-state kinetic and inhibition studies were performed for His6-PDK1(deltaPH)-catalyzed phosphorylation of PDK1-Tide (Tide), which contains an extended "PIF" sequence C-terminal to the consensus sequence for PDK1 phosphorylation. In two-substrate kinetics, a large degree of negative binding synergism was observed to occur on formation of the active ternary complex (alphaKd(ATP) = 40 microM and alphaKd(Tide) = 80 microM) from individual transitory binary complexes (Kd(ATP) = 0.6 microM and Kd(Tide) = 1 microM). On varying ATP concentrations, the ADP product and the (T/E)-PDK1-Tide product analog (p'Tide) behaved as competitive and noncompetitive inhibitors, respectively; on varying Tide concentrations, ADP and p'Tide behaved as noncompetitive and competitive inhibitors, respectively. Also, negative binding synergism was associated with formation of dead-end inhibited ternary complexes. Time progress curves in pre-steady-state studies under "saturating" or kcat conditions showed (i) no burst or lag phenomena, (ii) no change in reaction velocity when adenosine 5'-O-(thiotriphosphate) was used as a phosphate donor, and (iii) no change in reaction velocity on increasing relative microviscosity (0 < or = eta/eta0 < or = 3). Taken together, PDK1-catalyzed trans-phosphorylation of PDK1-Tide approximates a Rapid Equilibrium Random Bi Bi system, where motions in the central ternary complex are largely rate-determining.

Fused protein of δPKC activation loop and PDK1‐interacting fragment (δAL‐PIF) functions as a pseudosubstrate and an inhibitory molecule for PDK1 when expressed in cells

To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as deltaAL-PIF, and characterized its properties in cultured cells. deltaAL-PIF consists of two fused proteins of the protein kinase Cdelta (deltaPKC) activation loop (deltaAL) and PDK1-interacting fragment (PIF). The phosphorylation of deltaAL-PIF was detected with anti-deltaPKC phospho-Thr505-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing deltaAL-PIF, basal phosphorylation level at the activation loop of PKBalpha, deltaPKC and gammaPKC was reduced, compared with that in control cells, suggesting that deltaAL-PIF functions as an inhibitory molecule for PDK1. deltaAL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of deltaAL-PIF on gammaPKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing deltaAL-PIF for 3 days. These findings revealed that deltaAL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.

Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis

Activity of the serine-threonine protein kinase PINOID (PID) has been implicated in the asymmetrical localization of the membrane-associated PINFORMED (PIN) family of auxin transport facilitators. However, the means by which PID regulates PIN protein distribution is unknown. We have used recombinant PID protein to dissect the regulation of PID activity in vitro. We demonstrate that intramolecular PID autophosphorylation is required for the ability of PID to phosphorylate an exogenous substrate. PID-like mammalian AGC kinases act in a phosphorylation cascade initiated by the phospholipid-associated kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds to the C-terminal hydrophobic PDK1-interacting fragment (PIF) domain found in PDK1 substrates. We find that Arabidopsis PDK1 interacts with PID, and that transphosphorylation by PDK1 increases PID autophosphorylation. We show that a PID activation loop serine is required for PDK1-dependent PID phosphorylation. This activation is rapid and requires the PIF domain. Cell extracts from flowers and seedling shoots dramatically increase PID phosphorylation in a tissue-specific manner. A PID protein variant in which the PIF domain was mutated failed to be activated by the seedling shoot extracts. PID immunoprecipitated from Arabidopsis cells in which PDK1 expression was inhibited by RNAi showed a dramatic reduction in transphosphorylation of myelin basic protein substrate. These results indicate that AtPDK1 is a potent enhancer of PID activity and provide evidence that phospholipid signaling may play a role in the signaling processes controlling polar auxin transport.

The Na +/H + exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by 3-phosphoinositide-dependent protein kinase 1

The Na +/H + exchanger regulatory factor 2 (NHERF2/TKA-1/E3KARP) contains two PSD-95/Dlg/ZO-1 (PDZ) domains which interact with the PDZ docking motif (X–(S/T)–X–(V/L)) of proteins to mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. One of the PDZ domains of NHERF2 interacts specifically with the DSLL, DSFL, and DTRL motifs present at the carboxy-termini of the 2-adrenergic receptor, the platelet-derived growth factor receptor, and the cystic fibrosis transmembrane conductance regulator, respectively. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) also carries a putative PDZ-binding motif (D–S–F–L) at its carboxy tail, implicated in the specific interaction with NHERF2. There is a 3-phosphoinositide-dependent protein kinase 1 (PDK1) interacting fragment (PIF) in the tail of NHERF2. Using pull-down assays and co-transfection experiments, we demonstrated that the DSFL tail of SGK1 interacts with the first PDZ domain of NHERF2 and the PIF of NHERF2 binds to the PIF-binding pocket of PDK1 to form an SGK1–NHERF2–PDK1 complex. Formation of the protein complex promoted the phosphorylation and activation of SGK1 by PDK1. Thus, it was suggested that NHERF2 mediates the activation and phosphorylation of SGK1 by PDK1 through its first PDZ domain and PIF motif, as a novel SGK1 activation mechanism.

Mechanism of Phosphorylation of Protein Kinase B/Akt by a Constitutively Active 3-Phosphoinositide-dependent Protein Kinase-1

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA.

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP- and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF-binding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.

PDK1 acquires PDK2 activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2

Background: Protein kinase B (PKB) is activated by phosphorylation of Thr308 and of Ser473. Thr308 is phosphorylated by the 3-phosphoinositide-dependent protein kinase-1 (PDK1) but the identity of the kinase that phosphorylates Ser473 (provisionally termed PDK2) is unknown. Results: The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF). PIF is situated carboxy-terminal to the kinase domain of PRK2, and contains a consensus motif for phosphorylation by PDK2 similar to that found in PKBα, except that the residue equivalent to Ser473 is aspartic acid. Mutation of any of the conserved residues in the PDK2 motif of PIF prevented interaction of PIF with PDK1. Remarkably, interaction of PDK1 with PIF, or with a synthetic peptide encompassing the PDK2 consensus sequence of PIF, converted PDK1 from an enzyme that could phosphorylate only Thr308 of PKBα to one that phosphorylates both Thr308 and Ser473 of PKBα in a manner dependent on phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P 3). Furthermore, the interaction of PIF with PDK1 converted the PDK1 from a form that is not directly activated by PtdIns(3,4,5)P 3 to a form that is activated threefold by PtdIns(3,4,5)P 3. We have partially purified a kinase from brain extract that phosphorylates Ser473 of PKBα in a PtdIns(3,4,5)P 3-dependent manner and that is immunoprecipitated with PDK1 antibodies. Conclusions: PDK1 and PDK2 might be the same enzyme, the substrate specificity and activity of PDK1 being regulated through its interaction with another protein(s). PRK2 is a probable substrate for PDK1.