The protein kinase enzyme family plays a pivotal role in almost every aspect of cellular function, including cellular metabolism, division, proliferation, transcription, movement, and survival. Protein kinase A (PKA), whose activation is triggered by cyclic adenosine monophosphate (cAMP), is widely distributed in various systems and tissues throughout the body and highly related to pathogenesis and progression of various kinds of diseases. The inhibition of PKA activation is essential for the study of PKA functions. Protein kinase inhibitor peptide (PKI) is a potent, heat-stable, and specific PKA inhibitor. It has been demonstrated that PKI can block PKA-mediated phosphorylase activation. Since then, researchers have a lot of knowledge about PKI. PKI is considered to be the most effective and specific method to inhibit PKA and is widely used in related research. In this review, we will first introduce the knowledge on the activation of PKA and mechanisms related on the inhibitory effects of PKI on PKA. Then, we will compare PKI-mediated PKA inhibition vs. several popular methods of PKA inhibition.
The protein kinase enzyme family plays a pivotal role in almost every aspect of cellular function, including cellular metabolism, division, proliferation, transcription, movement, and survival. Protein kinases function by phosphorylating proteins, which is balanced with dephosphorylation by phosphoprotein phosphatases, making phosphorylation-dephosphorylation an effective regulatory process.
Protein kinase A (PKA) was the first protein kinase to be discovered. It was identified in skeletal muscle by Edmond H. Fisher and Edwin G. Krebs when they found that glycogen phosphorylase was activated following its phosphorylation by protein kinase, which was later termed “phosphorylase kinase” and now “protein kinase A”. Almost at the same time, Sutherland et al. demonstrated that cyclic adenosine monophosphate (cAMP), a heat-stable compound initially characterized in 1957 by Cook and the colleagues, led to the activation of PKA. As such, PKA is also termed “cAMP-dependent protein kinase.”
As a prototypical serine/threonine kinase, PKA is distributed in all systems and tissues throughout the body and related to pathogenesis and progression of various diseases. Numerous studies have focused on the function, regulation, and pathological roles of this signaling pathway. Manipulation of this signaling pathway has been explored to treat several diseases such as cardiovascular diseases, Alzheimer disease, Parkinson’s disease, ischemia, and diabetes.
The discovery of cAMP-mediated activation of PKA led to the “Second Messenger Hypothesis” in cellular signaling. It is described as that extracellular ligands, such as a certain kind of hormones like glucagon and epinephrine acted on membrane receptors and cause the generation of intracellular second messengers like cAMP, which in turn activated a kind of protein kinase and lead to the activation of cellular processes like glycogenolysis. cAMP is the first described second messenger, which generated by activated adenylyl cylase.
PKA is inactive in the absence of the triggering cAMP. Like most protein kinases, PKA was found in an inactive state under basal conditions and activated by a variety of ligand-induced regulatory mechanisms. The underlying molecular mechanism for the activation of PKA by cAMP intracellularly has been extensively studied and is the prototype of protein kinase activation. PKA is a tetrameric holoenzyme comprising two regulatory (R) and two catalytic (C) subunits. The C subunit has three isoforms Cα, Cβ, and Cγ, while the R subunit can be categorized into two subtypes (type I and II) according to their distribution, biochemical properties (e.g., sensitivities to cAMP), and affinity to A-kinase Anchoring Protein (AKAP). Each subtype of R subunits includes α and β isoforms. The R subunits serve three roles: binding to and inhibiting the C subunit and anchoring PKA to scaffolding protein AKAP. The RI subunit has a pseudosubstrate sequence, which has high affinity with the C subunit but cannot be phosphorylated by the C subunit, thus inhibiting the C subunit. The RII subunit is a substrate and also an inhibitor of the C subunit though phosphorylated RII does not dissociate from the C subunit in the absence of cAMP. In general, most AKAPs show more specificity to the RII subunit than to the RI subunit though D-AKAP1 and D-AKAP2 may have the same high affinity to RI and RII. RI has been found in cytosol and mitochondria. Currently more than 70 AKAPs have been identified. AKAP-mediated targeting of PKA to certain subcellular compartments provides the spatial and temporal regulation of the whole signaling cascade. In the presence of cAMP, each regulatory subunit binds to two molecules of cAMP at separate allosteric binding sites (one high affinity site and one low affinity site), leading to reduced affinity between the R and the C subunits and separation of the holoenzyme of PKA into a regulatory subunit dimer and two monomeric catalytic subunits. The released catalytic subunits become active and phosphorylate their substrates on serine and/or threonine sites in different signaling microdomains, leading to the conduction of cellular biological function.
PKA signaling orchestrated by AKAP plays critical roles in regulating cardiovascular, neuronal, and other functions and diseases. In the brain, an AKAP called Yotiao is anchored to the plasma membrane of neurons and scaffolds PKA and the NR1 subunit of post-synaptic N-methyl-D-aspartic acid (NMDA) receptor. When PKA is activated, it causes substantial phosphorylation and activation of NMDA, which plays a vital role in the regulation of synaptic plasticity. AKAP79/150 may anchor the L-type Ca 2+ channels to the membrane in a supermolecular complex to coordinate the regulation of the channel. WAVE1 (Wiskott-Alrich syndrome protein family verprolin homology protein 1), an AKAP, is associated with cytoskeleton to transduce and regulate basic cellular functions. Muscle specific AKAP (mAKAP) on the nuclear envelope may regulate PKA activity, which is critical for gene expression in the nucleus. AKAPs on the outer membrane of mitochondria regulate PKA-dependent phosphorylation of Bad to exert an antiapoptotic effect. One research group suggested that the released free PKA C subunits do not diffuse far away from the microdomains but this has been challenged. Nonetheless, activated PKA C has been shown to diffuse into the nucleus to phosphorylate nuclear proteins including transcription factors such as the cAMP-response element binding (CREB) protein.
The termination of PKA signaling includes multiple counteracting mechanisms: the degradation of cAMP by phosphodiesterases (PDEs), the dephosphorylation of PKA substrates by phosphatases, and the inhibition of PKA by PKI, a soluble peptide in the cell. While many studies have addressed the degradation of cAMP and the dephosphorylation of PKA substrates, the physiological and pathological roles of PKA inhibition by PKI remain to be elucidated. We will focus on the regulation of PKA by PKI in this review.
PKI is a heat-stable interfering peptide, firstly identified in skeletal muscle extract and which inhibited the activation of PKA. It has been demonstrated that PKI led to blocks the ability of cAMP to catalyze PKA activation. PKI is widely distributed in a variety of tissues including the brain, heart, liver, testes, skeletal muscles, and pancreas, and thus plays an important role in the pathogenesis and progression of cancer, neurodegeneration disorders, and drug addiction. There are three endogenous PKI isoforms, PKIα, β, and γ, which expressed in cell-specific and/or tissue-specific expression patterns. In the cell, it seems that all three isoforms are concentrated in the nucleus and at high level in the cytosol. PKIα is highly expressed in skeletal and cardiac muscles, brain, liver, pancreas, and expressed moderately in kidney, and colon; while PKIβ is highly expressed in the testis and moderately expressed in the spleen and cerebellum. PKIγ is highly expressed in the heart and moderately expressed in the brain, pancreas, lungs, liver, skeletal muscle, kidney, spleen, prostate, testis, ovary, small intestine, and colon.
