Sprouty proteins: modified modulators, matchmakers or missing links?

Sprouty proteins are involved in organogenesis, particularly during the branching of endothelial tubes, and existing evidence suggests that Sprouty's point of action lies downstream of receptor signaling to inhibit the activation of the central Ras/Erk pathway. How Sprouty proteins accomplish their inhibitory action and whether they interact with other signaling pathways are significant questions. Sprouty proteins are devoid of any recognizable protein interaction domain, and clues as to how they function have been mainly derived from screening for interacting partners. Conserved across all the Sprouty proteins are three sequences: a Cbl-tyrosine kinase-binding (TKB) binding motif centered on an obligatorily phosphorylated tyrosine (Y55 in Sprouty2), a serine-rich motif (SRM) and a cysteine-rich domain (CRD). With the exception of a handful of proteins that bind to the N-terminus, most of the binding to Sprouty occurs via the CRD, predominantly by serine/threonine kinases that target sites within the SRM on Sprouty. Some of the resultant increase in phosphorylation is opposed by activated protein phosphatase 2A that binds to the N-terminal Cbl-TKB binding motif. Significantly, two ubiquitin E3 ligases also bind to the N-terminus of Sprouty: c-Cbl binds with high affinity to the TKB binding motif and SIAH2 binds constitutively to a different site; both proteins are able to direct the ubiquitination of Sprouty proteins and its destruction. The collective evidence points to Sprouty proteins as being substantially covalently-modified to control its location, stability, association, and destruction. With such stringent control of the Sproutys, the main question is what key proteins does this facilitator bring together?

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Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels

eLife ◽

10.7554/elife.35222 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 17

Author(s):

Jacqueline Niu ◽

Ivy E Dick ◽

Wanjun Yang ◽

Moradeke A Bamgboye ◽

David T Yue ◽

...

Keyword(s):

Regulatory Subunit ◽

Global Changes ◽

Binding Motif ◽

Rich Domain ◽

Binding Interface ◽

Cellular Excitability ◽

Allosteric Sites ◽

Nav Channels ◽

Cam Regulation ◽

Cysteine Rich Domain

Calmodulin (CaM) serves as a pervasive regulatory subunit of CaV1, CaV2, and NaV1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca2+-feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (stac) and fibroblast growth factor homologous factors (fhf) selectively diminish Ca2+/CaM-regulation of CaV1 and NaV1 families, respectively. The two proteins operate on allosteric sites within upstream portions of respective channel carboxy-tails, distinct from the CaM-binding interface. Generalizing this mechanism, insertion of a short RxxK binding motif into CaV1.3 carboxy-tail confers synthetic switching of CaM regulation by Mona SH3 domain. Overall, our findings identify a general class of auxiliary proteins that modify Ca2+/CaM signaling to individual targets allowing spatial and temporal orchestration of feedback, and outline strategies for engineering Ca2+/CaM signaling to individual targets.

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The Linker Domain of SNAP25 Acts as a Flexible Molecular Spacer to Ensure Efficient S-Acylation

10.1101/2020.01.21.914333 ◽

2020 ◽

Author(s):

Christine Salaun ◽

Jennifer Greaves ◽

Nicholas C.O. Tomkinson ◽

Luke H. Chamberlain

Keyword(s):

Amino Acids ◽

Binding Motif ◽

Linear Motif ◽

Linker Region ◽

Rich Domain ◽

Enzyme Substrate ◽

Efficient Coupling ◽

Affinity Interaction ◽

Linker Domain ◽

Cysteine Rich Domain

ABSTRACTS-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the “mini-linker” region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, a SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.

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Activation by phosphorylation of phosphofructokinase from the annelid Lumbricus terrestris and comparison of phosphorylated sites in invertebrate phosphofructokinases

Biochemical Journal ◽

10.1042/bj3170377 ◽

1996 ◽

Vol 317 (2) ◽

pp. 377-383 ◽

Cited By ~ 7

Author(s):

Ralph SCHALOSKE ◽

Michael BIETHINGER ◽

Linda A. FOTHERGILL-GILMORE ◽

Hans Werner HOFER

Keyword(s):

Protein Phosphatase ◽

Helix Pomatia ◽

Lumbricus Terrestris ◽

Catalytic Subunit ◽

Dependent Protein Kinase ◽

N Terminus ◽

Phosphatase 2A ◽

Dependent Protein ◽

Earthworm Lumbricus

Purified phosphofructokinase from the earthworm Lumbricus terrestris was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase from the same organism to an extent of approx. 0.5 mol/mol of subunit. Activation of the enzyme occurred in parallel to the incorporation of covalently bound phosphate and was reversed by the action of the catalytic subunit of protein phosphatase 2A. Phosphorylation decreased the co-operativity of fructose 6-phosphate saturation in the presence of inhibitory concentrations of ATP, and increased the apparent Vmax obtained with saturating concentrations of the activators 5´-AMP and fructose 2,6-bisphosphate. The phosphorylated sites of phosphofructokinase from L. terrestris and from two molluscs (Helix pomatia and Mytilus edulis) were sequenced and shown to exhibit distinct similarity to sequences located near to the N-terminus of nematode phosphofructokinases [Klein, Olson, Favreau, Wintertowed, Hatzenbuhler, Shea, Nulf and Geary (1991) Mol. Biochem. Parasitol. 48, 17–26].

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A Direct Interaction between the N Terminus of Adenylyl Cyclase AC8 and the Catalytic Subunit of Protein Phosphatase 2A

Molecular Pharmacology ◽

10.1124/mol.105.018275 ◽

2005 ◽

Vol 69 (2) ◽

pp. 608-617 ◽

Cited By ~ 27

Author(s):

Andrew J. Crossthwaite ◽

Antonio Ciruela ◽

Timothy F. Rayner ◽

Dermot M. F. Cooper

Keyword(s):

Adenylyl Cyclase ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Direct Interaction ◽

Catalytic Subunit ◽

N Terminus ◽

Phosphatase 2A

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Lysine-Independent Turnover of Cyclin G1 Can Be Stabilized by B′α Subunits of Protein Phosphatase 2A

Molecular and Cellular Biology ◽

10.1128/mcb.00907-08 ◽

2008 ◽

Vol 29 (3) ◽

pp. 919-928 ◽

Cited By ~ 5

Author(s):

Hongyun Li ◽

Koji Okamoto ◽

Melissa J. Peart ◽

Carol Prives

Keyword(s):

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Protein Product ◽

Transcriptional Level ◽

Cyclin G1 ◽

N Terminus ◽

Lysine Residues ◽

Phosphatase 2A ◽

Cyclin G ◽

In Cells

ABSTRACT Although the cyclin G1 gene is known to be regulated at the transcriptional level by p53, less is understood about the turnover of its protein product. We found that ectopically and endogenously expressed cyclin G1 protein is highly unstable and is degraded by a proteasome-mediated pathway. The N-terminal 137 amino acids of cyclin G1 (cyclin G1-137) are necessary and sufficient for both cyclin G1 ubiquitination and turnover. Interestingly, a mutant cyclin G1 (8KR) in which all lysine residues in this region have been replaced with arginine can be both ubiquitinated in cells and stabilized by a proteasome inhibitor to a similar extent as wild-type cyclin G1-137. Furthermore, the presence of a six-Myc tag at the N terminus of cyclin G1-137 significantly inhibits the protein's turnover, suggesting a role for the extreme N terminus of the protein in ubiquitin-mediated proteolysis. Although we and others previously showed that cyclin G1 protein can bind to MDM2, which functions as an E3 ubiquitin ligase to p53 and itself, cyclin G1 protein can be degraded in cells without MDM2 and p53. Interestingly, the B′α1 subunit of the serine/threonine protein phosphatase 2A, which binds to cyclin G1, can stabilize cyclin G1 under unstressed conditions and upon DNA damage, as well as inhibit the ability of cyclin G1 to be ubiquitinated. Our results thus indicate that proteasomal turnover of cyclin G1 is regulated by noncanonical processes.

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Functional Multimerization of Human Telomerase Requires an RNA Interaction Domain in the N Terminus of the Catalytic Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.22.4.1253-1265.2002 ◽

2002 ◽

Vol 22 (4) ◽

pp. 1253-1265 ◽

Cited By ~ 74

Author(s):

Tara J. Moriarty ◽

Sylvain Huard ◽

Sophie Dupuis ◽

Chantal Autexier

Keyword(s):

Reverse Transcriptase ◽

Rna Binding ◽

Catalytic Subunit ◽

Binding Motif ◽

Telomerase Rna ◽

Human Telomerase Reverse Transcriptase ◽

Interaction Domain ◽

N Terminus ◽

Human Telomerase ◽

Rna Interaction

ABSTRACT Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase [hTERT]) containing reverse transcriptase (RT)-like motifs. The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms. We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization. Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity. We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif. Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70%. Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity. Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.

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A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment

eLife ◽

10.7554/elife.55966 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Xinru Wang ◽

Dimitriya H Garvanska ◽

Isha Nasa ◽

Yumi Ueki ◽

Gang Zhang ◽

...

Keyword(s):

Protein Phosphatase ◽

Electrostatic Interactions ◽

Binding Motif ◽

Dynamic Binding ◽

Dynamic Charge ◽

Charge Interaction ◽

Phosphatase 2A ◽

Fine Tune ◽

Positively Charged ◽

New Framework

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

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B56 Regulatory Subunit of Protein Phosphatase 2A Mediates Valproic Acid-Induced p300 Degradation

Molecular and Cellular Biology ◽

10.1128/mcb.25.2.525-532.2005 ◽

2005 ◽

Vol 25 (2) ◽

pp. 525-532 ◽

Cited By ~ 25

Author(s):

Jihong Chen ◽

Jonathan R. St-Germain ◽

Qiao Li

Keyword(s):

Valproic Acid ◽

Transcriptional Activation ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Regulatory Subunit ◽

Interaction Domain ◽

Regulatory Subunits ◽

Subunit Expression ◽

Phosphatase 2A ◽

The Stability

ABSTRACT Transcriptional coactivator p300 is required for embryonic development and cell proliferation. Valproic acid, a histone deacetylase inhibitor, is widely used in the therapy of epilepsy and bipolar disorder. However, it has intrinsic teratogenic activity through unidentified mechanisms. We report that valproic acid stimulates proteasome-dependent p300 degradation through augmentation of gene expression of the B56γ regulatory subunits of protein phosphatase 2A. The B56γ3 regulatory and catalytic subunits of protein phosphatase 2A interact with p300. Overexpression of the B56γ3 subunit leads to proteasome-mediated p300 degradation and represses p300-dependent transcriptional activation, which requires the B56γ3 interaction domain of p300. Conversely, silencing of the B56γ subunit expression by RNA interference increases the stability and transcriptional activity of the coactivator. Our study establishes the functional interaction between protein phosphatase 2A and p300 activity and provides direct evidence for signal-dependent control of p300 function.

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