Protein–protein interactions in plant mitogen-activated protein kinase cascades

Protein–protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein–protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein–protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α–Edr2 interaction was verified by co-localization analysis. Functionally, this p38α–Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α–MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)–MK5 and of the p38α–p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.

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Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2

Biochemical Journal ◽

10.1042/bj20031628 ◽

2004 ◽

Vol 380 (1) ◽

pp. 19-30 ◽

Cited By ~ 63

Author(s):

Guadalupe SABIO ◽

Suzana REUVER ◽

Carmen FEIJOO ◽

Masato HASEGAWA ◽

Gareth M. THOMAS ◽

...

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

Pdz Domain ◽

Mitogen Activated Protein Kinase ◽

Protein Protein Interactions ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

A Cell ◽

Cellular Stresses ◽

Sb 203580

SAPK3 (stress-activated protein kinase-3, also known as p38γ) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of α1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38γ and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38γ binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38γ to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38α and SAPK2b/p38β but not SAPK3/p38γ, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38γ with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38γ co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38γ, a first physiological substrate described for SAPK3/p38γ and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein–protein interactions at the synapse in response to adverse stress- or mitogenrelated stimuli.

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In silico and in vitro investigations on the protein–protein interactions of glutathione S-transferases with mitogen-activated protein kinase 8 and apoptosis signal-regulating kinase 1

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2020.1827036 ◽

2020 ◽

pp. 1-11

Author(s):

Chakradhara Rao S. Uppugunduri ◽

Jayaraman Muthukumaran ◽

Shannon Robin ◽

Teresa Santos-Silva ◽

Marc Ansari

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

In Silico ◽

Mitogen Activated Protein Kinase ◽

Protein Protein Interactions ◽

Mitogen Activated Protein ◽

Glutathione S Transferases

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An ultrasensitive fiveplex activity assay for cellular kinases

Scientific Reports ◽

10.1038/s41598-019-55998-8 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Christian M. Smolko ◽

Kevin A. Janes

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Large Scale ◽

Cell Types ◽

Mitogen Activated Protein Kinase ◽

Activity Measurement ◽

Kinase Substrate ◽

Iκb Kinase ◽

Mitogen Activated Protein

AbstractProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 µg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

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Identification of key microRNAs in the carotid arteries of ApoE−/− mice exposed to disturbed flow

Hereditas ◽

10.1186/s41065-019-0112-x ◽

2019 ◽

Vol 156 (1) ◽

Author(s):

Xinzhou Wang ◽

Shuibo Gao ◽

Liping Dai ◽

Zhentao Wang ◽

Hong Wu

Keyword(s):

Blood Flow ◽

Protein Interactions ◽

Carotid Arteries ◽

Target Genes ◽

Pathological Process ◽

Mitogen Activated Protein Kinase ◽

Molecular Pathways ◽

Protein Protein Interactions ◽

Mitogen Activated Protein ◽

Limma Package

Abstract Background Atherosclerosis (AS) is one of the main causes of cardiovascular disease. AS plaques often occur in blood vessels with oscillatory blood flow and their formation can be regulated by microRNAs (miRNAs). The aim of this study is to identify the key miRNAs and molecular pathways involved in this pathological process. Methods In this study, gene chip data obtained from the GEO database was analyzed using the LIMMA package to find differentially expressed miRNAs (DE miRNAs) in the carotid arteries of ApoE−/− mice exposed to different blood flow rates. Predicted targets of the DE miRNAs were identified using the TargetScan, miRDB, and DIANA databases respectively, and the potential target genes (PTGs) were found by analyzing the common results of three databases. The DAVID database was used to enrich the PTGs based on gene ontology (GO) and pathway (Kyoto Encyclopedia of Genes and Genomes, KEGG), and the STRING database was used to uncover any protein-protein interactions (PPI) of the PTGs. Results The networks of the DE miRNAs-PTGs, Pathway-PTGs-DE miRNAs, and PTGs PPI, were constructed using Cytoscape, and 11 up-regulated and 13 down-regulated DE miRNAs and 1479 PTGs were found. GO results showed that PTGs were significantly enriched in functions such as transcriptional regulation and DNA binding. KEGG results showed that PTGs were significantly enriched in inflammation-related mitogen-activated protein kinase (MAPK) and AS-related FOXO pathways. The PPI network revealed some key target genes in the PTGs. Conclusions The analysis of key miRNAs and molecular pathways that regulate the formation of AS plaques induced by oscillatory blood flow will provide new ideas for AS treatment.

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Signalling pathways and the regulation of SUMO modification

Biochemical Society Transactions ◽

10.1042/bst0351414 ◽

2007 ◽

Vol 35 (6) ◽

pp. 1414-1418 ◽

Cited By ~ 48

Author(s):

B. Guo ◽

S.-H. Yang ◽

J. Witty ◽

A.D. Sharrocks

Keyword(s):

Protein Interactions ◽

Molecular Mechanisms ◽

Mitogen Activated Protein Kinase ◽

Signalling Pathways ◽

Protein Protein Interactions ◽

E3 Ligases ◽

Sumo Modification ◽

Mitogen Activated Protein ◽

The Impact ◽

Protein Sumoylation

The modification of proteins by SUMO (small ubiquitin-related modifier) conjugation is becoming increasingly recognized as an important regulatory event. Protein SUMOylation can control a whole range of activities, including subcellular localization, protein–protein interactions and enzymatic activity. However, the SUMOylation process can itself be controlled. In the present review, the mechanisms through which protein SUMOylation is regulated are discussed, with particular emphasis on the impact of signalling pathways. A major point of regulation of the SUMO pathway is through targeting the E3 ligases, and a number of different ways to achieve this have been identified. More generally, the MAPK (mitogen-activated protein kinase) pathways represent one way through which SUMOylation of specific proteins is controlled, by using molecular mechanisms that at least in part also function by modifying the activity of SUMO E3 ligases. Further intricacies in signalling pathway interactions are hinted at through the growing number of examples of cross-talk between different post-translational modifications and SUMO modification.

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Identification of Constitutive and Ras-Inducible Phosphorylation Sites of KSR: Implications for 14-3-3 Binding, Mitogen-Activated Protein Kinase Binding, and KSR Overexpression

Molecular and Cellular Biology ◽

10.1128/mcb.19.1.229 ◽

1999 ◽

Vol 19 (1) ◽

pp. 229-240 ◽

Cited By ~ 146

Author(s):

Angela M. Cacace ◽

Neil R. Michaud ◽

Marc Therrien ◽

Karen Mathes ◽

Terry Copeland ◽

...

Keyword(s):

Signal Transduction ◽

Protein Kinase ◽

Protein Interactions ◽

Mek Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Ras Signaling ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Mitogen Activated Protein

ABSTRACT Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. InXenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.

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Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D

Blood ◽

10.1182/blood.v99.4.1434 ◽

2002 ◽

Vol 99 (4) ◽

pp. 1434-1441 ◽

Cited By ~ 20

Author(s):

Pamela J. Mansfield ◽

Vania Hinkovska-Galcheva ◽

Shannon S. Carey ◽

James A. Shayman ◽

Laurence A. Boxer

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Phospholipase D ◽

Protein Interactions ◽

Polymorphonuclear Leukocyte ◽

Cytochalasin B ◽

Adenosine Diphosphate ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein ◽

Oxidant Production

Exogenous C2-ceramide has been shown to inhibit polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of phospholipase D (PLD) and downstream events, including activation of extracellular signal–regulated kinases 1 and 2, leading to the hyphothesis that the sphingomyelinase pathway is involved in termination of phagocytosis. Here it is postulated that increased PLD activity generating phosphatidic acid and diacylglycerol (DAG) is essential for superoxide release and degranulation and that ceramide, previously shown to be generated during PMN activation, inhibits PLD activation, thereby leading to inhibition of PMN function. When PMNs were primed with granulocyte colony-stimulating factor (G-CSF) and then activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C2-ceramide (10 μM) completely inhibited release of superoxide, lactoferrin, and gelatinase; the DAG analog sn-1,2-didecanoylglycerol (DiC10) (10 μM) restored oxidase activation and degranulation in the ceramide-treated cells. Similarly, C2-ceramide inhibited oxidase activity and degranulation of PMNs treated with cytochalasin B followed by FMLP, and DiC10 restored function. In contrast, C2-ceramide did not inhibit phosphorylation of p47phox or p38 mitogen-activated protein kinase, or translocation of p47phox, PLD-containing organelles, adenosine diphosphate–ribosylation factor 1, RhoA, protein kinase C (PKC)–β or PKC-α to the plasma membrane in G-CSF or cytochalasin B–treated, FMLP-activated PMNs. PLD activity increased by 3-fold in G-CSF–primed PMNs stimulated by FMLP and by 30-fold in cytochalasin B–treated PMNs stimulated by FMLP. Both PLD activities were completely inhibited by 10 μM C2-ceramide. In conclusion, superoxide, gelatinase, and lactoferrin release require activation of the PLD pathway in primed PMNs and cytochalasin B–treated PMNs. Ceramide may affect protein interactions with PLD in the plasma membrane, thereby attenuating PMN activation.

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An ultrasensitive fiveplex activity assay for cellular kinases

10.1101/687715 ◽

2019 ◽

Author(s):

Christian M. Smolko ◽

Kevin A. Janes

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Large Scale ◽

Cell Types ◽

Mitogen Activated Protein Kinase ◽

Activity Measurement ◽

Kinase Substrate ◽

Iκb Kinase ◽

Mitogen Activated Protein

ABSTRACTProtein kinases are enzymes whose abundance, protein-protein interactions, and posttranslational modifications together determine net signaling activity in cells. Large-scale data on cellular kinase activity are limited, because existing assays are cumbersome, poorly sensitive, low throughput, and restricted to measuring one kinase at a time. Here, we surmount the conventional hurdles of activity measurement with a multiplexing approach that leverages the selectivity of individual kinase-substrate pairs. We demonstrate proof of concept by designing an assay that jointly measures activity of five pleiotropic signaling kinases: Akt, IκB kinase (IKK), c-jun N-terminal kinase (JNK), mitogen-activated protein kinase (MAPK)-extracellular regulated kinase kinase (MEK), and MAPK-activated protein kinase-2 (MK2). The assay operates in a 96-well format and specifically measures endogenous kinase activation with coefficients of variation less than 20%. Multiplex tracking of kinase-substrate pairs reduces input requirements by 25-fold, with ~75 μg of cellular extract sufficient for fiveplex activity profiling. We applied the assay to monitor kinase signaling during coxsackievirus B3 infection of two different host-cell types and identified multiple differences in pathway dynamics and coordination that warrant future study. Because the Akt–IKK–JNK–MEK–MK2 pathways regulate many important cellular functions, the fiveplex assay should find applications in inflammation, environmental-stress, and cancer research.

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Expression and Purification of Active Human Kinases Using Pichia pastoris as A General-Purpose Host

10.21203/rs.2.17140/v2 ◽

2020 ◽

Author(s):

May H. Abdel Aziz ◽

Yao Fan ◽

Lijun Liu ◽

Mark Moasser ◽

Haian Fu ◽

...

Keyword(s):

Protein Kinase ◽

Pichia Pastoris ◽

Protein Interactions ◽

Active Form ◽

Methylotrophic Yeast ◽

Aurora Kinase ◽

General Purpose ◽

Mitogen Activated Protein Kinase ◽

Methylotrophic Yeast Pichia Pastoris ◽

Mitogen Activated Protein

Abstract Background: The heterologous expression of human kinases in good purity and in a monomeric, soluble and active form can be challenging. Most of the reported successful attempts are carried out in insect cells as a host. The use of E. coli for expression is limited to a few kinases and usually is facilitated by large solubility tags that can limit biophysical studies and affect protein–protein interactions. In this report, we evaluate the methylotrophic yeast Pichia pastoris (P. pastoris) as a general-purpose host for expression of human kinases. Methods: Six diverse kinases were chosen due to their therapeutic importance in human cancers. Tested proteins include serine/threonine kinases cyclin-dependent kinases 4 and 6 (CDK4 and 6) and aurora kinase A (AurKA), receptor tyrosine kinase erbB-2 (HER2), and dual specificity kinase mitogen-activated protein kinase kinase 3 (MKK3b). Noting that positively charged kinases expressed with higher yield, we sought to improve expression of two challenging targets, CDK6 and HER2, by fusing the highly basic, N-terminal domain of the secreted tyrosine-protein kinase VLK. A standard expression procedure was developed for P. pastoris, followed by purification using affinity chromatography. Purity and activity of the proteins were confirmed and compared to published values. Results: Some kinases were purified with good yield and purity and with comparable activity to commercially available versions. Addition of the VLK domain improved expression and decreased aggregation of CDK6 and HER2. Conclusions: P. pastoris is a promising host for expression of soluble and active human kinases.

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