The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome

Although HEF1 has a well-defined role in integrin-dependent attachment signalling at focal adhesions, it relocalizes to the spindle asters at mitosis. We report here that overexpression of HEF1 causes an increase in centrosome numbers and multipolar spindles, resembling defects induced by manipulation of the mitotic regulatory kinase Aurora-A (AurA). We show that HEF1 associates with and controls activation of AurA. We also show that HEF1 depletion causes centrosomal splitting, mono-astral spindles and hyperactivation of Nek2, implying additional action earlier in the cell cycle. These results provide new insight into the role of an adhesion protein in coordination of cell attachment and division.     

Inhibitors of angiogenesis and cancer-related receptor tyrosine kinases

Inhibition of tumor angiogenesis is an attractive target in cancer therapy. In this context, receptor tyrosine kinases play a pivotal role. Extensive efforts have been made to identify and develop small-molecule inhibitors of these central signaling proteins. Some of these compounds have already passed or are currently in clinical trials to investigate their applicability as anti-cancer drugs. However, the high expectations that are set in antiangiogenic therapy have not yet been accomplished. But there are also new and exciting opportunities for cancer treatment by combining antiangiogenic molecules with newly emerging therapeutics.     

A translational regulator, PUM2, promotes both protein stability and kinase activity of Aurora-A

Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/C(Cdh1). Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/C(Cdh1)-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis.     

Structures of thermophilic and mesophilic adenylate kinases from the genus Methanococcus

The crystal structures of adenylate kinases from the thermophile Methanococcus thermolithotrophicus and the mesophile Methanococcus voltae have been solved to resolutions of 2.8A and 2.5A, respectively. The structures of the enzymes are similar to that of the adenylate kinase from archaeal Sulfolobus acidocaldarius in many respects such as the extended central beta-sheets, the short LID domain, and the trimeric state. The analysis of unligated and AMP-bound subunits of M.voltae suggests that movements of two mobile domains are not independent of each other. The methanococcal structures are examined with respect to their lack of the "invariant" Lys residue within the phosphate-binding loop, and two Arg residues in the LID domain are proposed as substituting residues based on their conservation among archaeal adenylate kinases and mobility within the structures. Since S.acidocaldarius adenylate kinase has the invariant Lys residue as well as the two Arg residues, its phosphate-binding loop is examined and compared with those of other adenylate kinases. On the basis of the comparison and other available biochemical data, the unusual conformation of the Lys residue in S.acidocaldarius adenylate kinase is explained. Despite possessing 78% sequence identity, the methanococcal enzymes exhibit significantly different thermal stabilities. To study the determinants of thermostability, several structural features including salt-links, hydrogen bonds, packing density, surface to volume ratio and buried surface area are compared between the enzymes. From their difference in apolar buried surface area, hydrophobic interaction is proposed to be a basis for the disparate thermostabilities, and the corresponding free energy difference is also estimated. Results of previous mutational studies are interpreted in terms of the crystal structures, and support the importance of hydrophobic interactions in thermostability.     

Structures of proteins and cofactors: X-ray crystallography

Protein crystallography is the predominately used technique for the determination of the three-dimensional structures of proteins and other macromolecules. In this article, the methodology utilized for the measurement and analysis of the diffraction data from crystals is briefly reviewed. As examples of both the usefulness and difficulties of this technique, the determination of the structures of several photosynthetic pigment–protein complexes is described, namely, the reaction center from purple bacteria, photosystem I and photosystem II from cyanobacteria, the light-harvesting complex II from purple bacteria, and the FMO protein from green bacteria.     

Optimization of a novel kinase inhibitor scaffold for the dual inhibition of JAK2 and FAK kinases

The elaboration of a novel scaffold for the inhibition of JAK2 and FAK kinases was targeted in order to provide a dual inhibitor that could target divergent pathways for tumor cell progression.     

Characterization of the tyrosine kinases RAFTK/Pyk2 and FAK in nerve growth factor-induced neuronal differentiation

The related adhesion focal tyrosine kinase (RAFTK), a member of the focal adhesion kinase (FAK) family and highly expressed in brain, is a key mediator of various extracellular signals that elevate intracellular Ca(2+) concentration. We investigated RAFTK and FAK signaling upon nerve growth factor (NGF) stimulation of PC12 cells. NGF induced the tyrosine phosphorylation of RAFTK in a time- and dose-dependent manner, whereas no change in the tyrosine phosphorylation of FAK was observed. Chemical inhibition showed that RAFTK phosphorylation was inhibited by blocking phospholipase Cgamma activity or intracellular Ca(2+). Blocking of extracellular Ca(2+) or phosphatidylinositol 3-kinase activity partially reduced the phosphorylation of RAFTK. In addition, disruption of actin polymerization abolished RAFTK phosphorylation, indicating that an intact actin-based cytoskeletal organization is required for RAFTK phosphorylation. The focal adhesion molecule paxillin was co-immunoprecipitated with RAFTK, and its tyrosine phosphorylation was increased in a Ca(2+)-dependent manner upon NGF stimulation. Confocal microscopic analysis demonstrated that RAFTK translocated from the cytoplasm to potential neurite initiation sites at the cell periphery, where RAFTK co-localized with paxillin and bundled actin in the early phase (within 5 min) of NGF stimulation, whereas FAK co-localized with paxillin at "point contacts," which are the primary cell adhesion sites in neuronal cells. Significant distribution of RAFTK was observed in the neurites and growth cones of differentiated PC12 cells. Furthermore, potassium depolarization induced the tyrosine phosphorylation of both RAFTK and paxillin in an intracellular Ca(2+)-dependent manner in the differentiated PC12 cells. Taken together, these results demonstrate that RAFTK is involved in NGF-induced cytoskeletal organization and may play a role in neurite and growth cone function(s).     

Phosphorylation, protein kinases and ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disease characterized by renal cyst formation and caused by mutations in the PKD1 and PKD2 genes, which encode polycystin-1(PC-1) and -2 (PC-2) proteins, respectively. PC-1 is a large plasma membrane receptor involved in the regulation of several biological functions and signaling pathways including the Wnt cascade, AP-1, PI3kinase/Akt, GSK3β, STAT6, Calcineurin/NFAT and the ERK and mTOR cascades. PC-2 is a calcium channel of the TRP family. The two proteins form a functional complex and prevent cyst formation, but the precise mechanism(s) involved remains unknown. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues

The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin αvβ3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCγ in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MΦs) treated with tumor necrosis factor-α (TNFα) or interleukin-1β (IL1β). RA and OA STs showed a greater percentage of pFAK on lining cells and MΦs compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFα or IL1β stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MΦs. pPyk2 was expressed on RA ST fibrobasts but not in MΦs at baseline, however it was upregulated upon TNFα or IL1β activation in both cell types. pSrc was expressed in RA ST fibroblasts and MΦs at baseline and was further increased by TNFα or IL1β stimulation. pPaxillin and pPLCγ were upregulated in RA versus OA and ND lining cells and sublining MΦs. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.     

Overcoming the challenges of membrane protein crystallography

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future.     

Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

Mitogen-activated protein (MAP) kinases bind tightly to many of their physiologically relevant substrates. We have identified a new subfamily of murine serine/threonine kinases, whose members, MAP kinase-interacting kinase 1 (Mnk1) and Mnk2, bind tightly to the growth factor-regulated MAP kinases, Erk1 and Erk2. MNK1, but not Mnk2, also binds strongly to the stress-activated kinase, p38. MNK1 complexes more strongly with inactive than active Erk, implying that Mnk and Erk may dissociate after mitogen stimulation. Erk and p38 phosphorylate MNK1 and Mnk2, which stimulates their in vitro kinase activity toward a substrate, eukaryotic initiation factor-4E (eIF-4E). Initiation factor eIF-4E is a regulatory phosphoprotein whose phosphorylation is increased by insulin in an Erk-dependent manner. In vitro, MNK1 rapidly phosphorylates eIF-4E at the physiologically relevant site, Ser209. In cells, Mnk1 is post-translationally modified and enzymatically activated in response to treatment with either peptide growth factors, phorbol esters, anisomycin or UV. Mitogen- and stress-mediated MNK1 activation is blocked by inhibitors of MAP kinase kinase 1 (Mkk1) and p38, demonstrating that Mnk1 is downstream of multiple MAP kinases. MNK1 may define a convergence point between the growth factor-activated and one of the stress-activated protein kinase cascades and is a candidate to phosphorylate eIF-4E in cells.     

Small molecule inhibitors of the Pyk2 and FAK kinases modulate chemoattractant-induced migration, adhesion and Akt activation in follicular and marginal zone B cells

B-lymphocytes produce protective antibodies but also contribute to autoimmunity. In particular, marginal zone (MZ) B cells recognize both microbial components and self-antigens. B cell trafficking is critical for B cell activation and is controlled by chemoattactants such as CXCL13 and sphingosine 1-phosphate (S1P). The related tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase (Pyk2) regulate cell migration and adhesion but their roles in B cells are not fully understood. Using a novel Pyk2-selective inhibitor described herein (PF-719), as well as a FAK-selective inhibitor, we show that both Pyk2 and FAK are important for CXCL13- and S1P-induced migration of B-2 cells and MZ B cells. In contrast, LFA-1-mediated adhesion required only Pyk2 whereas activation of the Akt pro-survival kinase required FAK but not Pyk2. Thus Pyk2 and FAK mediate critical processes in B cells and these inhibitors can be used to further elucidate their functions in B cells.     

Characterization of protein kinases from adrenal medulla

Since phosphorylation of chromosomal proteins by cyclic AMP-dependent protein kinases (EC 2.7.1.37) enhances template activity of adrenal medulla chromatin (9), we have studied the properties and regulation of protein kinases isolated from chromaffin cell cytosol and nuclei. DEAE-cellulose chromatography revealed three peaks of kinase activity in the nucleus (nPKI, nPKII, nPKIII) and two in the cytosol (cPKI, cPKII). The three nuclear enzymes, as well as cPKII, did not require cyclic AMP to express their catalytic activity, nPKI and nPKIII preferred acidic substrates as PO ₄ ^3– acceptors, while nPKII and the cytosol enzymes preferred basic PO ₄ ^3– acceptors. Enzyme recombination experiments using protein kinase regulatory subunits from cytosol suggested that cPKII was the catalytic subunit of cPKI. In contrast, the nuclear enzymes were not catalytic subunits of the cyclic AMP-dependent protein kinase in the cytosol (cPKI). Only the cytosol protein kinases could be inhibited by endogenous heat-stable protein kinase inhibitors. The nuclear and cytosol cyclic AMP-independent protein kinases were distinguishable on the basis of their sedimentation constants as well as Mg²⁺ and Mn²⁺ requirements.     

Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA

Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC–MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins.     

Conservation, variability and the modeling of active protein kinases

The human proteome is rich with protein kinases, and this richness has made the kinase of crucial importance in initiating and maintaining cell behavior. Elucidating cell signaling networks and manipulating their components to understand and alter behavior require well designed inhibitors. These inhibitors are needed in culture to cause and study network perturbations, and the same compounds can be used as drugs to treat disease. Understanding the structural biology of protein kinases in detail, including their commonalities, differences and modes of substrate interaction, is necessary for designing high quality inhibitors that will be of true use for cell biology and disease therapy. To this end, we here report on a structural analysis of all available active-conformation protein kinases, discussing residue conservation, the novel features of such conservation, unique properties of atypical kinases and variability in the context of substrate binding. We also demonstrate how this information can be used for structure prediction. Our findings will be of use not only in understanding protein kinase function and evolution, but they highlight the flaws inherent in kinase drug design as commonly practiced and dictate an appropriate strategy for the sophisticated design of specific inhibitors for use in the laboratory and disease therapy.     

Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases

The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66-74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.     

NKIAMRE,a novel conserved CDC2-related kinase with features of both mitogen-activated protein kinases and cyclin-dependent kinases

We report the cloning of the NKIAMRE gene located on human chromosome 5q31.1. It encodes a novel 52kDa Cdc2-related kinase with a 1.5kb open reading frame. Like MAP kinases, NKIAMRE contains a Thr-X-Tyr (TXY) motif in the activation loop domain. Similar to cdks, NKIAMRE contains the putative negative regulatory Ser14 and Tyr15 residues and the cyclin-binding motif, NKIAMRE, from which it derives its name. Human NKIAMRE has significant amino acid identity to related kinases in rat, mouse, Caenorhabditis elegans, and Drosophila, and is widely expressed in human tissues and cell lines. Confocal microscopy demonstrates that NKIAMRE localizes to the cytoplasm. NKIAMRE is activated by treatment of cells with phorbol 12-myristate 13-acetate. Mutation of the ATP-binding Lys-33 to arginine and the Thr-Glu-Tyr motif to Ala-Glu-Phe abolished its ability to phosphorylate myelin basic protein. NKIAMRE is a member of a conserved family of kinases with homology to both MAP kinases and cyclin-dependent kinases.     

Identification of the soluble EphA7-interacting protein Nicalin as a regulator of EphA7 expression

Molecular and Cellular Biochemistry - A soluble form of EphA7 (sEphA7) has been found to antagonize the role of full-length EphA7 (EphA7-FL) to stabilize the membrane level of the tight junction...