Claude Cochet

A Structural Basis for Substrate Specificities of Protein Ser/Thr Kinases: Primary Sequence Preference of Casein Kinases I and II, NIMA, Phosphorylase Kinase, Calmodulin- Dependent Kinase II, CDK5, and Erk1

We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.

Joining the cell survival squad: an emerging role for protein kinase CK2

Protein kinase CK2 (formerly known as casein kinase 2) is a serine/threonine protein kinase whose functions have been under investigation for over three decades, leading to the recognition that it interacts with several signaling pathways. Recent demonstrations of signal-mediated dynamic localization of CK2 and the identification of new signaling targets for it have converged to indicate an unexpected function for this protein kinase: cell survival. Here, we summarize our emerging knowledge about how CK2 might participate in the transduction of survival signals.

Disruption of the Regulatory β Subunit of Protein Kinase CK2 in Mice Leads to a Cell-Autonomous Defect and Early Embryonic Lethality

ABSTRACT Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory β subunit, CK2β, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2β gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2β−/− morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2β is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2β. Thus, our study demonstrates that in mammals, CK2β is essential for viability at the cellular level, possibly because it acquired new functions during evolution.

Fibroblast growth factor-2 binds to the regulatory beta subunit of CK2 and directly stimulates CK2 activity toward nucleolin.

The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory β subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory β subunit.

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger‐mediated dimerization

Protein kinase CK2 is a tetramer composed of two α catalytic subunits and two β regulatory subunits. The structure of a C‐terminal truncated form of the human β subunit has been determined by X‐ray crystallography to 1.7 Å resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely α‐helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among β subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits.

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

Identification of Polyoxometalates as Nanomolar Noncompetitive Inhibitors of Protein Kinase CK2

Protein kinase CK2 is a multifunctional kinase of medical importance that is dysregulated in many cancers. In this study, polyoxometalates were identified as original CK2 inhibitors. [P2Mo18O62](6-) has the most potent activity. It inhibits the kinase in the nanomolar range by targeting key structural elements located outside the ATP- and peptide substrate-binding sites. Several polyoxometalate derivatives exhibit strong inhibitory efficiency, with IC50 values < or = 10 nM. Furthermore, these inorganic compounds show a striking specificity for CK2 when tested in a panel of 29 kinases. Therefore, polyoxometalates are effective CK2 inhibitors in terms of both efficiency and selectivity and represent nonclassical kinase inhibitors that interact with CK2 in a unique way. This binding mode may provide an exploitable mechanism for developing potent drugs with desirable properties, such as enhanced selectivity relative to ATP-mimetic inhibitors.

Selectivity, Cocrystal Structures, and Neuroprotective Properties of Leucettines, a Family of Protein Kinase Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimers disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimers disease.

Phosphorylation by CK2 and MAPK enhances calnexin association with ribosomes

Calnexin was initially identified as an endoplasmic reticulum (ER) type I integral membrane protein, phosphorylated on its cytosolic domain by ER‐associated protein kinases. Although the role of the ER luminal domain of calnexin has been established as a constituent of the molecular chaperone machinery of the ER, less is known about the role of the cytosolic phosphorylation of calnexin. Analysis by two‐dimensional phosphopeptide maps revealed that calnexin was in vitro phosphorylated in isolated microsomes by casein kinase 2 (CK2) and extracellular‐signal regulated kinase‐1 (ERK‐1) at sites corresponding to those for in vivo phosphorylation. In canine pancreatic microsomes, synergistic phosphorylation by CK2 and ERK‐1 led to increased association of calnexin with membrane‐bound ribosomes. In vivo, calnexin‐associated ERK‐1 activity was identified by co‐immunoprecipitation. This activity was abolished in cells expressing a dominant‐negative MEK‐1. Activation of ERK‐1 in cells by addition of serum led to a 4‐fold increase in ribosome‐associated calnexin over unstimulated cells. Taken together with studies revealing calnexin association with CK2 and ERK‐1, a model is proposed whereby phosphorylation of calnexin leads to a potential increase in glycoprotein folding close to the translocon.

Polyamine-mediated protein phosphorylations: a possible target for intracellular polyamine action.

Polyamines are well-known ubiquitous components of living cells. Although these polycations have been implicated in the regulation of major cellular functions such as DNA, RNA and protein synthesis occurring during cellular proliferation and/or differentiation processes, their mechanism of action at the molecular level has remained obscure. On the other hand, protein phosphorylation has emerged as a regulatory process of prime importance in cellular regulation. Data have recently been presented suggesting that polyamines may express at least part of their biological action through an effect upon selective protein phosphorylation systems. Two types of polyamine-sensitive protein kinases have been characterized in the last few years. The best known in molecular terms is the widespread casein kinase G (also termed casein kinase II), which represents a multifunctional protein kinase, at present classified as a messenger-independent activity. The other is a polyamine-dependent nuclear ornithine decarboxylase kinase characterized in Physarum polycephalum and several mammalian tissues. Both protein kinases are activated by polyamines in vitro at concentrations compatible with a physiological role, by a mechanism which most likely also involves an effect through the protein substrate conformation. Preliminary evidence suggests that both kinases may be implicated in the regulation of DNA-dependent RNA polymerase activities, although several other potential substrates have been suggested for casein kinase G. Another suggestion is that these kinases may also participate in the post-translational regulation of ornithine decarboxylase, the rate-limiting step in the polyamine biosynthetic pathway. A novel class of protein kinase activities may thus be defined as polyamine-mediated phosphorylation systems for which polyamines may function as intracellular messenger. Although their biological significance remains to be fully established, especially with regard to the definition of their specific intracellular target(s) and subsequent biological functions, these systems will be interesting to consider in future studies aimed at understanding the role of polyamines in cell regulation.