Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1

Self Cite

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by 32 P i revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of "docking interactions." Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2-4).3-Phosphoinositide-dependent protein kinase 1 (PDK1) 3 belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum-and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6 -9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10). Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulator...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

Dettori

Sonzogni

Meyer

et al. 2009

Self Cite

“…Although the most likely reason is the relatively low affinity of these unoptimized scaffolds, one cannot exclude the possibility that these chemotypes do not modulate the PDK1 functional activity despite their specific binding. At the present time, because of the low affinity of the allosteric ligands, both those discovered in this work and those described in the literature (15,16,37,38), any meaningful modulation studies in intact cells are deferred until more potent ligands are developed from these initial leads in the future.…”

Section: Discussionmentioning

confidence: 99%

“…Several independent studies demonstrated that the PIF pocket is amenable to small molecule binding (15,16,37,38). Using a virtual screening approach of a ϳ60,000 compound chemical library, Engel et al (15) identified several compounds that increased the activity of ⌬PH-PDK1.…”

Section: Discussionmentioning

confidence: 99%

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

Bobkova

Weber

et al. 2010

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 M 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 1؋ and 10؋ 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.Protein kinases are the second largest group of drug targets after G-protein-coupled receptors. They account for an estimated 20 -30% of drug development pipelines and comprise the largest enzyme family, with more than 500 members encoded in the human genome (1, 2). It is estimated that protein kinases catalyze the reversible phosphorylation of more than 10,000 substrate proteins at greater than 100,000 sites (3, 4). Consequently, malfunctioning of kinases can have profound effects on human health, and the discovery of selective kinase inhibitors is critical in helping to delineate the role of these enzymes in disease processes.In cancer, oncogenic transformations are frequently associated with increased activity of protein-serine/threonine kinases, many of which are key signaling molecules in the phosphoinositide 3-kinase and mitogen-activated protein kinase (MAPK) 2 pathways. Recent clinical studies have demonstrated that activating mutations in the MAPK pathway (i.e. KRAS and BRAF) confer resistance to anti-epidermal growth factor receptor antibody therapy (5, 6), suggesting that co-targeting of receptor tyrosine kinases and downstream serine/threonine kinases may be an attractive therapeutic strategy.The serine/threonine pr...

“…There is increasing precedent for development of allosteric kinase inhibitors, which have proven to be much more selective than classical ATP competitive inhibitors (21,(43)(44)(45)(46)(47)(48)(49). Allosteric modulation of kinases is particularly promising because many soluble kinases have inter-or intradomain associations that allosterically regulate kinase activity.…”

Section: Discussionmentioning

confidence: 99%

Biophysical and Mechanistic Insights into Novel Allosteric Inhibitor of Spleen Tyrosine Kinase

Hall

Aulabaugh

Rajamohan

et al. 2012

Background: Spleen tyrosine kinase (Syk) is important for antigenic and inflammation immune responses. Results: These studies focus on activation and allosteric inhibition of Syk by a novel small molecule. Conclusion: We show Syk activation involves structural elongation, whereas allosteric inhibition results in contraction. Significance: We propose the allosteric inhibitor acts by reinforcing natural intramolecular regulation in Syk that normally keeps its kinase activity quiescent.