Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

Lateral diffusion plays a crucial role in numerous processes that take place in cell membranes, yet it is quite poorly understood in native membranes characterized by, e.g., domain formation and large concentration of proteins. In this article, we use atomistic and coarse-grained simulations to consider how packing of membranes and crowding with proteins affect the lateral dynamics of lipids and membrane proteins. We find that both packing and protein crowding have a profound effect on lateral diffusion, slowing it down. Anomalous diffusion is observed to be an inherent property in both protein-free and protein-rich membranes, and the time scales of anomalous diffusion and the exponent associated with anomalous diffusion are found to strongly depend on packing and crowding. Crowding with proteins also has a striking effect on the decay rate of dynamical correlations associated with lateral single-particle motion, as the transition from anomalous to normal diffusion is found to take place at macroscopic time scales: while in protein-poor conditions normal diffusion is typically observed in hundreds of nanoseconds, in protein-rich conditions the onset of normal diffusion is tens of microseconds, and in the most crowded systems as large as milliseconds. The computational challenge which results from these time scales is not easy to deal with, not even in coarse-grained simulations. We also briefly discuss the physical limits of protein motion. Our results suggest that protein concentration is anything but constant in the plane of cell membranes. Instead, it is strongly dependent on proteins' preference for aggregation.

Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using unbiased molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivity of V617F, the predominant JAK2 MPN mutation.

The JAK-STAT signal transduction pathway is responsible for mediating signals of over fifty cytokines, growth factors and hormones. Signaling through the JAK-STAT pathway is regulated on multiple levels, including intramolecular regulation by the JAK pseudokinase domain, and intermolecular regulation by a host of regulatory proteins. The advent of accessible genomic tools have provided a wealth of information on disease-associated mutations in the JAK-STAT pathway and its regulatory components. The vast number of these mutations in diseases ranging from immunodeficiencies and obesity to many cancers highlight the importance of correct regulation of JAK-STAT signaling for biological processes such as hematopoiesis, regulation of the immune system, metabolism, and growth. Simultaneously, JAK inhibitors are gaining traction in clinical use, both for treatment of diseases driven by JAK mutations, and for a host of inflammatory disorders, in which proinflammatory cytokine signaling through the JAK-STAT pathway is an integral part of pathogenesis. The elucidation of molecular mechanisms in the pathogenesis of complex diseases has also, however, brought the limitations of our current understanding on the regulation of cytokine signaling to the foreground. Indeed, deeper understanding of these regulatory mechanisms are a prerequisite for the development of the next generation of pharmacological modulators of the JAK-STAT pathway. In this review we discuss the current state of knowledge of the intra- and intermolecular regulation of the JAK-STAT pathway, with a focus on diseases arising from disruptions in the regulatory apparatus.

We describe how membrane proteins diffuse laterally in the membrane plane together with the lipids surrounding them. We find a number of intriguing phenomena. The lateral displacements of the protein and the lipids are strongly correlated, as the protein and the neighboring lipids form a dynamical protein-lipid complex, consisting of approximately 50-100 lipids. The diffusion of the lipids in the complex is much slower compared to the rest of the lipids. We also find a strong directional correlation between the movements of the protein and the lipids in its vicinity. The results imply that in crowded membrane environments there are no "free" lipids, as they are all influenced by the protein structure and dynamics. Our results indicate that, in studies of cell membranes, protein and lipid dynamics have to be considered together.