Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Abstract

The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...

The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα

“…One intriguing explanation is that the regulatory-catalytic domain interaction, which is significantly stronger in the presence of nucleotide, physically prevents phosphatases from accessing the phosphorylation sites on the catalytic domain. Finally, we recently observed that both PKC and PKA show positive cooperativity between substrate and ATP binding to the catalytic domain (34), which highlights a potentially conserved mechanism that drives catalytic-regulatory domain interactions.…”

Bitopic Inhibition of ATP and Substrate Binding in Ser/Thr Kinases through a Conserved Allosteric Mechanism

“…We recently demonstrated that BimI, a staurosporine derivative, inhibits binding of both ATP and substrate peptides to the PKC α catalytic domain. 9 In this work, FRET measurements of a range of nucleotide and staurosporine analogues reveal a systematic correlation between inhibitor structure and substrate displacement. Combining FRET measurements with MD simulation analysis, we uncover an allosteric switch region located outside the ATP binding site.…”

“…8,9 We have previously demonstrated that the potent ATP-competitive PKC inhibitor BimI displaces substrate binding to the catalytic domain of PKC α . 9 To test a potential intersection between BimI and the allosteric switch mechanism involving K347−D481, we used MD simulations to examine the conformational dynamics of PKC α in the presence of BimI. The amine group at the end of the n -propyl chain of BimI makes a salt bridge with D481 to disrupt the D481−K347 interaction (Figure 2A).…”

“…The SPASM sensors detect the strength of the PKC α −EGFR substrate peptide interaction using FRET. 9 Displacement of substrate binding results in a decrease in the intensity of the FRET signal relative to that of the apo state (negative Δ FRET). We have previously established that changes in the FRET intensity ratio ( Δ FRET) correlate linearly with the mole fraction of kinase bound to the substrate within the sensor.…”

IKBKE inhibits TSC1 to activate the mTOR/S6K pathway for oncogenic transformation

“…that their interaction could be transient, as in the case of many other similar interactions involving a kinase and its substrate (Sommese and Sivaramakrishnan, 2016). Next, we tested if TSC1 is a direct substrate for IKKε by performing an in vitro kinase assay ( Figure 4C).…”