Reciprocal Activation by Cyclin-Dependent Kinases 2 and 7 Is Directed by Substrate Specificity Determinants outside the T Loop

ABSTRACT Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

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A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5214 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5214-5225 ◽

Cited By ~ 137

Author(s):

A D Catling ◽

H J Schaeffer ◽

C W Reuter ◽

G R Reddy ◽

M J Weber

Keyword(s):

Protein Interactions ◽

Critical Role ◽

Phosphorylation Site ◽

Specific Protein ◽

Protein Protein Interactions ◽

Serum Stimulation ◽

And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

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The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0379 ◽

2016 ◽

Vol 27 (24) ◽

pp. 3841-3854 ◽

Cited By ~ 13

Author(s):

Felicitas Rataj ◽

Séverine Planel ◽

Agnès Desroches-Castan ◽

Juliette Le Douce ◽

Khadija Lamribet ◽

...

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

Mrna Decay ◽

Rna Binding ◽

Phosphorylation Site ◽

P38 Map Kinase ◽

Site Directed Mutagenesis ◽

Protein Protein Interactions

TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3′-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein–protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Biomolecules ◽

10.3390/biom11040510 ◽

2021 ◽

Vol 11 (4) ◽

pp. 510

Author(s):

Maho Yamamoto ◽

Rina Kondo ◽

Haruka Hozumi ◽

Seita Doi ◽

Miwako Denda ◽

...

Keyword(s):

Protein Interactions ◽

Biochemical Characterization ◽

S100 Protein ◽

High Mobility ◽

Dependent Manner ◽

Protein Signaling ◽

Protein Protein Interactions ◽

High Mobility Group Protein ◽

Pulldown Assay

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

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Ways into Understanding HIF Inhibition

Cancers ◽

10.3390/cancers13010159 ◽

2021 ◽

Vol 13 (1) ◽

pp. 159

Author(s):

Tina Schönberger ◽

Joachim Fandrey ◽

Katrin Prost-Fingerle

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Protein Protein Interactions ◽

Low Oxygen ◽

Drug Candidates ◽

Open Questions ◽

Wide Range ◽

Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

British Journal of Cancer ◽

10.1038/s41416-020-01250-4 ◽

2021 ◽

Author(s):

Liqing Jia ◽

Xiaolu Ge ◽

Chao Du ◽

Linna Chen ◽

Yanhong Zhou ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Protein Interactions ◽

Elongation Factor ◽

Protein Translation ◽

Protein Protein Interactions ◽

Promising Target ◽

Tgfβ Receptor ◽

Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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Multiple Interactions within the AKAP220 Signaling Complex Contribute to Protein Phosphatase 1 Regulation

Journal of Biological Chemistry ◽

10.1074/jbc.m010398200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 12128-12134 ◽

Cited By ~ 41

Author(s):

Robynn V. Schillace ◽

James W. Voltz ◽

Alistair T. R. Sim ◽

Shirish Shenolikar ◽

John D. Scott

Keyword(s):

Intermolecular Interactions ◽

Protein Interactions ◽

Protein Phosphatase ◽

Competitive Inhibitor ◽

Regulatory Subunit ◽

Protein Protein Interactions ◽

Signaling Complex ◽

Activity Measurements ◽

Anchoring Protein ◽

Pka Regulatory Subunit

The phosphorylation status of cellular proteins is controlled by the opposing actions of protein kinases and phosphatases. Compartmentalization of these enzymes is critical for spatial and temporal control of these phosphorylation/dephosphorylation events. We previously reported that a 220-kDa A-kinase anchoring protein (AKAP220) coordinates the location of the cAMP-dependent protein kinase (PKA) and the type 1 protein phosphatase catalytic subunit (PP1c) (Schillace, R. V., and Scott, J. D. (1999)Curr. Biol.9, 321–324). We now demonstrate that an AKAP220 fragment is a competitive inhibitor of PP1c activity (Ki= 2.9 ± 0.7 μm). Mapping studies and activity measurements indicate that several protein-protein interactions act synergistically to inhibit PP1. A consensus targeting motif, between residues 1195 and 1198 (Lys-Val-Gln-Phe), binds but does not affect enzyme activity, whereas determinants between residues 1711 and 1901 inhibit the phosphatase. Analysis of truncated PP1c and chimeric PP1/2A catalytic subunits suggests that AKAP220 inhibits the phosphatase in a manner distinct from all known PP1 inhibitors and toxins. Intermolecular interactions within the AKAP220 signaling complex further contribute to PP1 inhibition as addition of the PKA regulatory subunit (RII) enhances phosphatase inhibition. These experiments indicate that regulation of PP1 activity by AKAP220 involves a complex network of intra- and intermolecular interactions.

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c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6021-6029.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6021-6029

Author(s):

R Metz ◽

A J Bannister ◽

J A Sutherland ◽

C Hagemeier ◽

E C O'Rourke ◽

...

Keyword(s):

Protein Interactions ◽

Transcriptional Activation ◽

Binding Protein ◽

Tata Box ◽

Binding Motif ◽

Protein Protein Interactions ◽

High Concentrations ◽

Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

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