SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein–protein interactions

The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)–MO25 interaction (as in the LKB1–STRADα–MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly cerebral cavernous malformations.

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Multiple regulatory domains on the Byr2 protein kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5876 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5876-5887 ◽

Cited By ~ 59

Author(s):

H Tu ◽

M Barr ◽

D L Dong ◽

M Wigler

Keyword(s):

Protein Kinase ◽

Catalytic Domain ◽

Intramolecular Interaction ◽

Mitogen Activated Protein Kinase ◽

Regulatory Domain ◽

Regulatory Domains ◽

Kinase Catalytic Domain ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

G Protein Alpha Subunit

Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.

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A Glance into MTHFR Deficiency at a Molecular Level

International Journal of Molecular Sciences ◽

10.3390/ijms23010167 ◽

2021 ◽

Vol 23 (1) ◽

pp. 167

Author(s):

Castrense Savojardo ◽

Giulia Babbi ◽

Davide Baldazzi ◽

Pier Luigi Martelli ◽

Rita Casadio

Keyword(s):

Protein Structure ◽

Catalytic Domain ◽

Markov Models ◽

Consensus Method ◽

Regulatory Domain ◽

Functional Protein ◽

Regulatory Domains ◽

Interaction Sites ◽

Protein Architecture ◽

Mthfr Deficiency

MTHFR deficiency still deserves an investigation to associate the phenotype to protein structure variations. To this aim, considering the MTHFR wild type protein structure, with a catalytic and a regulatory domain and taking advantage of state-of-the-art computational tools, we explore the properties of 72 missense variations known to be disease associated. By computing the thermodynamic ΔΔG change according to a consensus method that we recently introduced, we find that 61% of the disease-related variations destabilize the protein, are present both in the catalytic and regulatory domain and correspond to known biochemical deficiencies. The propensity of solvent accessible residues to be involved in protein-protein interaction sites indicates that most of the interacting residues are located in the regulatory domain, and that only three of them, located at the interface of the functional protein homodimer, are both disease-related and destabilizing. Finally, we compute the protein architecture with Hidden Markov Models, one from Pfam for the catalytic domain and the second computed in house for the regulatory domain. We show that patterns of disease-associated, physicochemical variation types, both in the catalytic and regulatory domains, are unique for the MTHFR deficiency when mapped into the protein architecture.

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Structural snapshots of RAF kinase interactions

Biochemical Society Transactions ◽

10.1042/bst20170528 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1393-1406 ◽

Cited By ~ 10

Author(s):

Soheila Rezaei Adariani ◽

Marcel Buchholzer ◽

Mohammad Akbarzadeh ◽

Saeideh Nakhaei-Rad ◽

Radovan Dvorsky ◽

...

Keyword(s):

Conformational Changes ◽

Protein Interactions ◽

Membrane Lipids ◽

Control Cell ◽

Mitogen Activated Protein Kinase ◽

Regulatory Domains ◽

Raf Kinase ◽

Rich Domain ◽

Molecular Events ◽

Mitogen Activated Protein

RAF (rapidly accelerated fibrosarcoma) Ser/Thr kinases (ARAF, BRAF, and CRAF) link the RAS (rat sarcoma) protein family with the MAPK (mitogen-activated protein kinase) pathway and control cell growth, differentiation, development, aging, and tumorigenesis. Their activity is specifically modulated by protein–protein interactions, post-translational modifications, and conformational changes in specific spatiotemporal patterns via various upstream regulators, including the kinases, phosphatase, GTPases, and scaffold and modulator proteins. Dephosphorylation of Ser-259 (CRAF numbering) and dissociation of 14-3-3 release the RAF regulatory domains RAS-binding domain and cysteine-rich domain for interaction with RAS-GTP and membrane lipids. This, in turn, results in RAF phosphorylation at Ser-621 and 14-3-3 reassociation, followed by its dimerization and ultimately substrate binding and phosphorylation. This review focuses on structural understanding of how distinct binding partners trigger a cascade of molecular events that induces RAF kinase activation.

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Role of Curing Temperature of Poly(Glycerol Sebacate) Substrates on Protein-Cell Interaction and Early Cell Adhesion

Polymers ◽

10.3390/polym13030382 ◽

2021 ◽

Vol 13 (3) ◽

pp. 382

Author(s):

Rubén Martín-Cabezuelo ◽

José Carlos Rodríguez-Hernández ◽

Guillermo Vilariño-Feltrer ◽

Ana Vallés-Lluch

Keyword(s):

Protein Interactions ◽

Chemical Properties ◽

Focal Adhesions ◽

Collagen Type I ◽

Inhibitory Effect ◽

Synthesis Temperature ◽

Curing Temperature ◽

Cell Protein ◽

Preliminary Treatment ◽

Type I

A novel procedure to obtain smooth, continuous polymeric surfaces from poly(glycerol sebacate) (PGS) has been developed with the spin-coating technique. This method proves useful for separating the effect of the chemistry and morphology of the networks (that can be obtained by varying the synthesis parameters) on cell-protein-substrate interactions from that of structural variables. Solutions of the PGS pre-polymer can be spin-coated, to then be cured. Curing under variable temperatures has been shown to lead to PGS networks with different chemical properties and topographies, conditioning their use as a biomaterial. Particularly, higher synthesis temperatures yield denser networks with fewer polar terminal groups available on the surface. Material-protein interactions were characterised by using extracellular matrix proteins such as fibronectin (Fn) and collagen type I (Col I), to unveil the biological interface profile of PGS substrates. To that end, atomic force microscopy (AFM) images and quantification of protein adsorbed in single, sequential and competitive protein incubations were used. Results reveal that Fn is adsorbed in the form of clusters, while Col I forms a characteristic fibrillar network. Fn has an inhibitory effect when incubated prior to Col I. Human umbilical endothelial cells (HUVECs) were also cultured on PGS surfaces to reveal the effect of synthesis temperature on cell behaviour. To this effect, early focal adhesions (FAs) were analysed using immunofluorescence techniques. In light of the results, 130 °C seems to be the optimal curing temperature since a preliminary treatment with Col I or a Fn:Col I solution facilitates the formation of early focal adhesions and growth of HUVECs.

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Structure of human dual-specificity phosphatase 7, a potential cancer drug target

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x1500504x ◽

2015 ◽

Vol 71 (6) ◽

pp. 650-656 ◽

Cited By ~ 4

Author(s):

George T. Lountos ◽

Brian P. Austin ◽

Joseph E. Tropea ◽

David S. Waugh

Keyword(s):

Catalytic Domain ◽

Three Dimensional ◽

Mitogen Activated Protein Kinase ◽

Cancer Drug ◽

Dual Specificity Phosphatase ◽

Dual Specificity ◽

Starting Point ◽

Mitogen Activated Protein ◽

Dual Specificity Phosphatases

Human dual-specificity phosphatase 7 (DUSP7/Pyst2) is a 320-residue protein that belongs to the mitogen-activated protein kinase phosphatase (MKP) subfamily of dual-specificity phosphatases. Although its precise biological function is still not fully understood, previous reports have demonstrated that DUSP7 is overexpressed in myeloid leukemia and other malignancies. Therefore, there is interest in developing DUSP7 inhibitors as potential therapeutic agents, especially for cancer. Here, the purification, crystallization and structure determination of the catalytic domain of DUSP7 (Ser141–Ser289/C232S) at 1.67 Å resolution are reported. The structure described here provides a starting point for structure-assisted inhibitor-design efforts and adds to the growing knowledge base of three-dimensional structures of the dual-specificity phosphatase family.

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Effects of inhibitors of protein kinase C and calpain in experimental delayed cerebral vasospasm

Journal of Neurosurgery ◽

10.3171/jns.1992.76.1.0111 ◽

1992 ◽

Vol 76 (1) ◽

pp. 111-118 ◽

Cited By ~ 48

Author(s):

Nobutaka Minami ◽

Eiichi Tani ◽

Yukio Maeda ◽

Ikuya Yamaura ◽

Masahiro Fukami

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Catalytic Domain ◽

Limited Proteolysis ◽

Maximum Effect ◽

Regulatory Domain ◽

Calphostin C ◽

Delayed Cerebral Vasospasm ◽

Kinase C ◽

Basilar Arteries

✓ Vasospasm was produced in adult mongrel dogs by a two-hemorrhage method, and the spastic basilar arteries were exposed via the transclival route on Day 7. Tonic contraction was produced in the normal canine basilar arteries by a local application of KCl or serotonin after transclival exposure. The exposed spastic and tonic basilar arteries then received a topical application of the following: 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7), a potent inhibitor of protein kinase C acting at the catalytic domain; calphostin C, a specific inhibitor of protein kinase C acting at the regulatory domain; or calpeptin, a selective inhibitor of calpain. Both spastic and tonic basilar arteries were effectively dilated by H-7. Calphostin C caused only slight dilation of spastic basilar arteries but moderate dilation of tonic basilar arteries. Dilation in response to calpeptin was remarkable in the spastic basilar arteries but slight in the tonic basilar arteries. The doses of calphostin C and calpeptin required to obtain maximum effect were markedly lower in the tonic model than in the spastic model. The spastic and tonic models had a similar dose-dependent response to H-7 but quite a different response to calphostin C or calpeptin, suggesting a difference in the function of protein kinase C and calpain in the two models. Furthermore, the effect of calphostin C on the reversal of vasospasm was increased significantly after topical treatment with calpeptin. It is suggested that the majority of the catalytic domain of protein kinase C is dissociated from the regulatory domain, probably by a limited proteolysis with calpain, and is markedly activated in vasospasm.

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Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly

Journal of Cell Science ◽

10.1242/jcs.109.10.2499 ◽

1996 ◽

Vol 109 (10) ◽

pp. 2499-2508 ◽

Cited By ~ 1

Author(s):

J.E. Murphy-Ullrich ◽

M.A. Pallero ◽

N. Boerth ◽

J.A. Greenwood ◽

T.M. Lincoln ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

Focal Adhesion ◽

Kinase Activity ◽

Catalytic Domain ◽

Focal Adhesions ◽

Matrix Proteins ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein

Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

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A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

10.21203/rs.3.rs-114803/v1 ◽

2020 ◽

Author(s):

Santana Royan ◽

Bernard Gutmann ◽

Catherine Colas des Francs-Small ◽

Suvi Honkanen ◽

Jason Schmidberger ◽

...

Keyword(s):

Rna Editing ◽

Protein Interactions ◽

Rna Binding ◽

Catalytic Domain ◽

Rna Binding Proteins ◽

Cytidine Deaminase ◽

Land Plant ◽

Target Sequence ◽

Pentatricopeptide Repeat

Abstract Targeted cytidine to uridine RNA editing is a widespread phenomenon throughout the land plant lineage. Members of the pentatricopeptide repeat (PPR) protein family act as the specificity factors in this process. These proteins consist of helix-turn-helix domains, each of which recognises a single RNA nucleotide following a well-elucidated code. A cytidine deaminase-like domain (present at the C-terminus of some PPR editing factors or provided in trans via protein-protein interactions) is the catalytic domain in the process. The huge expansion of the PPR superfamily in land plants provides the sequence variation required for design of novel consensus-based RNA-binding proteins. We used this approach to construct a synthetic RNA editing factor designed to target one of the two sites in the Arabidopsis chloroplast transcriptome naturally recognised by the RNA editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that this designed editing factor specifically recognises the target sequence in in vitro binding assays and can partially complement a clb19 mutant. The designed factor is specific for the target rpoA site and does not recognise or edit the other site recognised by CLB19 in the clpP1 transcript. We show that the designed editing factor can function equally specifically in the bacterium E. coli, and shows some activity even in the absence of the editing cofactors that are often required for natural editing factor activity in plants. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

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The LxVP and PxIxIT NFAT Motifs Bind Jointly to Overlapping Epitopes on Calcineurin’s Catalytic Domain Distant to the Regulatory Domain

Structure ◽

10.1016/j.str.2014.05.006 ◽

2014 ◽

Vol 22 (7) ◽

pp. 1016-1027 ◽

Cited By ~ 9

Author(s):

Maayan Gal ◽

Shuai Li ◽

Rafael E. Luna ◽

Koh Takeuchi ◽

Gerhard Wagner

Keyword(s):

Catalytic Domain ◽

Regulatory Domain

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A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0157 ◽

2011 ◽

Vol 22 (14) ◽

pp. 2509-2519 ◽

Cited By ~ 44

Author(s):

Jian J. Liu ◽

Rebecca A. Stockton ◽

Alexandre R. Gingras ◽

Ararat J. Ablooglu ◽

Jaewon Han ◽

...

Keyword(s):

Endothelial Cell ◽

Small Gtpases ◽

Cell Junctions ◽

Physical Interaction ◽

Dependent Manner ◽

Cardiovascular Development ◽

Cavernous Malformations ◽

Cell Cell

Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

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