Edmond Fischer's kinase legacy: History of the protein kinase inhibitor and protein kinase A

Abstract

Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

Keywords: Edmond Fischer; kinase; protein kinase A; protein kinase inhibitor; pseudo-substrate; small linear motifs.

Figure 1.. Alignment of conserved motifs in the protein kinase superfamily.

The sequence of PKA, published in 1981 was aligned with the cloned sequence of Src . This alignment confirmed that Src and the PKA C-subunit had evolved from a common precursor and established unambiguously that Src and PKA belonged to the same gene family. Based on the alignment of 25 protein kinases, Quin et al aligned 25 protein kinase sequences and identified a set of 11 sub-domains that each contained conserved motifs .

Figure 2.. Structures of the PKA C-subunit bound to IP20 and ATP.

The first PKA structure solved in 1991 was a binary complex (left panel) that contained the PKA C-subunit and IP20 ^, . When excess Mg⁺⁺ was added, the ternary complex was obtained . The p−3 to p+1 Inhibitor Site reaches across the active site cleft (black box) while the distal amphipathic helix docks onto a hydrophobic surface of the C-subunit as a hydrophobic tethering site (red box).

Figure 3.. Small Linear Motifs embedded in PKI.

There are several Small Linear Motifs (SLiMs) embedded in the sequences of the three PKI isoforms. The first motif that docks onto the PKA C-subunit is bi-functional and includes the amphipathic helix, a beta-turn, and the Inhibitor sequences as indicated in Table 1. The second SLiM corresponds to an amphipathic helix that docks onto CRM1:Ran(GTP) ^, .

Figure 4.. Capturing Order/Disorder transitions of PKI.

The middle panel, based on NMR , captures the disorder of the inhibitor site of IP20 in the absence of the PKA C-subunit, while in the presence of the PKA C-subunits and ATP(Mg)₂ the inhibitor site is docked into the active site cleft of the bilobal C-subunit (white ribbon). In contrast the helix, which is partially ordered in solution, is firmly anchored to a hydrophobic pocket on the surface of the PKA C-subunit. On the left and right are models of the length PKI in the absence (left) and presence (right) of the PKA C-subunit. These models best represent the SAXS data. The overall ordered docking of full-length PKI, based on NMR, SAXS, fluorescence, meta-dynamics and Markov State modeling, provides our first mechanistic model for the dynamic docking process and also shows how the region following the Inhibitor Site is not only primed for recognition of CRM1 but also how it mediates allosteric cross-talk with the C-lobe of the PKR C-subunit .

Figure 5.. Binding of the PKA C-subunit and CREM1:Ran(GTP) to PKI.

The two Slim motifs in PKI bind with high-affinity to the PKA C-subunit (PDB:1ATP) and CRM1:Ran(GTP) (PDB:3NBY). In both cases high-affinity binding is mediated by the hydrophobic surface of an amphipathic helix.

Figure 6.. Distal tethering sites on the C-Lobe of the PKA C-subunit trap high affinity inhibitor proteins.

All PKA inhibitor proteins, PKI isoforms and R-subunits, share a common Inhibitor Site. PKI (left) and RI-subunit (middle) are pseudo-substrates and require ATP(Mg)₂, while RII-subunits are substrates and require only the two cyclic nucleotide binding (CNB) domain tethering sites (CNB-A and CNB-B) to achieve high-affinity binding. Left panel: High-affinity binding of IP20 requires the amphipathic helix containing Phe10 that lies N-terminal to the inhibitor site while the Inhibitor site is trapped by ATP(Mg)2 . The middle panel shows how the CNB-A domain of RIα is docked onto the C-lobe of the kinase via a different distal tethering site while the inhibitor site is trapped by ATP(Mg)₂. In this case high-affinity binding also requires the region that lies N-terminal to the Inhibitor site, but it is distinct from the PKI tethering site. The right panel shows how the RIIα subunit docks onto the CNB-A tethering site and onto the CNB-B tethering site and this is sufficient to convey high-affinity binding in the absence of ATP .

Figure 7.. Tissue-specific expression of PKA and PKI isoforms.

The expression taken from the GEXT web server shows that PKIα is highly enriched in the brain. Cα (PRKACA), RIα (PRKAR1A), and RIIα (PRKAR2A) are expressed constitutively at high levels in most cells while the expression of Cβ (PRKACB), RIβ (PRKAR1B), and RIIβ (PRKAR2B) are all enriched in neuronal tissues.

Figure 8.. Imaging of endogenous PKI and PKA subunits.

Left panel: High-Resolution Mosaic Imaging of RIβ (red) and RIIβ (green) in mouse hippocampus using isoform-specific antibodies . Right Panel: Imaging of PKIα mRNA in dentate gyrus region of rat hippocampus . Panels A and C compare the hemisphere that had been injected with kainic acid to stimulate synaptic activity (C) with the untreated hemisphere (A). Panels B and D compare electrolytic lesion of the hilius, which is known to stimulate synaptic input to granule cells.