Kinases Phosphatases, Volume 1, Issue 4 (December 2023) – 5 articles

The structural knowledge about protein kinase CK2 is dominated by crystal structures of human CK2α, the catalytic subunit of human CK2, and the product of the CSNK2A1 gene. In contrast, far fewer structures of CK2α′, its paralogous isoform and the product of the CSNK2A2 gene, have been published. However, according to a PDB survey, CK2α′ is the superior alternative for crystallographic studies because of the inherent potential of the single mutant CK2α′^Cys336Ser to provide crystal structures with atomic resolution. In particular, a triclinic crystal form of CK2α′^Cys336Ser is a robust tool to determine high-quality enzyme-ligand complex structures via soaking. In this work, further high-resolution CK2α′^Cys336Ser structures in complex with selected ligands emphasizing this trend are described. In one of these structures, the “N-terminal segment site”, a small-molecule binding region never found in any eukaryotic protein kinase and holding the potential for the development of highly selective substrate-competitive CK2 inhibitors, was discovered. In order to also address the binding site for the non-catalytic subunit CK2β, which is inaccessible in these triclinic CK2α′^Cys336Ser crystals for small molecules, a reliable path to a promising monoclinic crystal form of CK2α′^Cys336Ser is presented. In summary, the quality of CK2α′^Cys336Ser as an exquisite crystallographic tool is solidified. Full article

Protein kinase casein kinase 2 (CK2/CSNK2) is a pleiotropic kinase involved in many cellular processes and, accordingly, has been identified as a potential target for therapeutic intervention for multiple indications. Significant research effort has been invested into identifying CK2 inhibitors as potential drug candidates and potent and selective CK2 chemical probes to interrogate CK2 function. Here, we review the small molecule inhibitors reported for CK2 and discuss various orthosteric, allosteric, and bivalent inhibitors of CK2. We focus on the pyrazolo[1,5-a]pyrimidines and naphthyridines, two chemotypes that have been extensively explored for chemical probe development. We highlight the uptake and demonstrated utility of the pyrazolo[1,5-a]pyrimidine chemical probe SGC-CK2-1 by the scientific community in cellular studies. Finally, we propose criteria for an ideal in vivo chemical probe for investigating CK2 function in a living organism. While no compound currently meets these metrics, we discuss ongoing and future directions in the development of in vivo chemical probes for CK2. Full article

Protein kinase A (PKA) signaling exemplifies phosphorylation-based signaling as we understand it today. Its catalytic-subunit structure and dynamics continue to advance our understanding of kinase mechanics as the first protein kinase catalytic domain to be identified, sequenced, cloned, and structurally detailed. The PKA holoenzyme elaborates on the role of its regulatory subunits and maintains our understanding of cAMP-dependent cellular signaling. The activation of PKA holoenzymes by cAMP is an example of specialized protein allostery, emphasizing the relevance of protein binding interfaces, unstructured regions, isoform diversity, and dynamics-based allostery. This review provides the most up-to-date overview of PKA structure and function, including a description of the catalytic and regulatory subunits’ structures. In addition, the structure, activation, and allostery of holoenzymes are covered. Full article

Protein kinase CK2 (formerly known as casein kinase 2 or II), a ubiquitous and constitutively active enzyme, is widely recognized as one of the most pleiotropic serine/threonine kinases. It plays a critical role in numerous signaling pathways, with hundreds of bona fide substrates. However, despite considerable research efforts, our understanding of the entire CK2 substratome and its functional associations with the majority of these substrates is far from being completely deciphered. In this context, we aim to provide an overview of how CK2 recognizes its substrates. We will discuss the pros and cons of the existing methods to manipulate CK2 activity in cells, as well as exploring the dynamic response of substrate phosphorylation to CK2 modulation. Full article

Receptor tyrosine kinase (RTK) KIT is key regulator of cellular signalling, and its deregulation contributes to the development and progression of many serious diseases. Several mutations lead to the constitutive activation of the cytoplasmic domain of KIT, causing the aberrant intracellular signalling observed in malignant tumours. Elucidating the molecular basis of mutation-induced effects at the atomistic level is absolutely required. We report the first dynamic 3D model (DYNASOME) of the full-length cytoplasmic domain of the oncogenic mutant KIT^D816V generated through unbiased long-timescale MD simulations under conditions mimicking the natural environment of KIT. The comparison of the structural and dynamical properties of multidomain KIT^D816V with those of wild type KIT (KIT^WT) allowed us to evaluate the impact of the D816V mutation on each protein domain, including multifunctional well-ordered and intrinsically disordered (ID) regions. The two proteins were compared in terms of free energy landscape and intramolecular coupling. The increased intrinsic disorder and gain of coupling within each domain and between distant domains in KIT^D816V demonstrate its inherent self-regulated constitutive activation. The search for pockets revealed novel allosteric pockets (POCKETOME) in each protein, KIT^D816V and KIT^WT. These pockets open an avenue for the development of new highly selective allosteric modulators specific to KIT^D816V. Full article