scholarly journals The first DEP domain of the RhoGEF P-Rex1 autoinhibits activity and contributes to membrane binding

2020 ◽  
Vol 295 (36) ◽  
pp. 12635-12647
Author(s):  
Sandeep K. Ravala ◽  
Jesse B. Hopkins ◽  
Caroline B. Plescia ◽  
Samantha R. Allgood ◽  
Madison A. Kane ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Phosphorylation Site ◽  
Solution Conformation ◽  
Domain Swap ◽  
Rac Gtpases ◽  
Highly Correlated ◽  
Charged Membranes ◽  
Dh Domain ◽  
Basic Loop ◽  
Kinase A

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) catalyzes the exchange of GDP for GTP on Rac GTPases, thereby triggering changes in the actin cytoskeleton and in transcription. Its overexpression is highly correlated with the metastasis of certain cancers. P-Rex1 recruitment to the plasma membrane and its activity are regulated via interactions with heterotrimeric Gβγ subunits, PIP3, and protein kinase A (PKA). Deletion analysis has further shown that domains C-terminal to its catalytic Dbl homology (DH) domain confer autoinhibition. Among these, the first dishevelled, Egl-10, and pleckstrin domain (DEP1) remains to be structurally characterized. DEP1 also harbors the primary PKA phosphorylation site, suggesting that an improved understanding of this region could substantially increase our knowledge of P-Rex1 signaling and open the door to new selective chemotherapeutics. Here we show that the DEP1 domain alone can autoinhibit activity in context of the DH/PH-DEP1 fragment of P-Rex1 and interacts with the DH/PH domains in solution. The 3.1 Å crystal structure of DEP1 features a domain swap, similar to that observed previously in the Dvl2 DEP domain, involving an exposed basic loop that contains the PKA site. Using purified proteins, we show that although DEP1 phosphorylation has no effect on the activity or solution conformation of the DH/PH-DEP1 fragment, it inhibits binding of the DEP1 domain to liposomes containing phosphatidic acid. Thus, we propose that PKA phosphorylation of the DEP1 domain hampers P-Rex1 binding to negatively charged membranes in cells, freeing the DEP1 domain to associate with and inhibit the DH/PH module.

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization

2021 ◽  
pp. 107732
Author(s):  
Nicolás González Bardeci ◽  
Enzo Tofolón ◽  
Felipe Trajtenberg ◽  
Julio Caramelo ◽  
Nicole Larrieux ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Regulatory Subunit ◽  
Kinase A

scholarly journals Generation of an Isogenic Collection of Yeast Actin Mutants and Identification of Three Interrelated Phenotypes

Genetics ◽  
2001 ◽  
Vol 157 (2) ◽  
pp. 533-543
Author(s):  
Johanna L Whitacre ◽  
Dana A Davis ◽  
Kurt A Toenjes ◽  
Sharon M Brower ◽  
Alison E M Adams
Keyword(s):  
Crystal Structure ◽  
Growth Rates ◽  
Small Region ◽  
Wild Type ◽  
Large Collection ◽  
Growth Defects ◽  
Cytoskeletal Function ◽  
Highly Correlated ◽  
The Relationship ◽  
Mutant Cells

Abstract A large collection of yeast actin mutations has been previously isolated and used in numerous studies of actin cytoskeletal function. However, the various mutations have been in congenic, rather than isogenic, backgrounds, making it difficult to compare the subtle phenotypes that are characteristic of these mutants. We have therefore placed 27 mutations in an isogenic background. We used a subset of these mutants to compare the degree to which different actin alleles are defective in sporulation, endocytosis, and growth on NaCl-containing media. We found that the three phenotypes are highly correlated. The correlations are specific and not merely a reflection of general growth defects, because the phenotypes are not correlated with growth rates under normal conditions. Significantly, those actin mutants exhibiting the most severe phenotypes in all three processes have altered residues that cluster to a small region of the actin crystal structure previously defined as the fimbrin (Sac6p)-binding site. We examined the relationship between endocytosis and growth on salt and found that shifting wild-type or actin mutant cells to high salt reduces the rate of α-factor internalization. These results suggest that actin mutants may be unable to grow on salt because of additive endocytic defects (due to mutation and salt).

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites

1994 ◽  
Vol 14 (1) ◽  
pp. 10-20
Author(s):  
M Wu ◽  
C D Allis ◽  
M T Sweet ◽  
R G Cook ◽  
T H Thatcher ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
High Mobility ◽  
Single Copy ◽  
Amino Acid Sequences ◽  
Evolutionary Analysis ◽  
Associated Proteins ◽  
Kinase Phosphorylation ◽  
Kinase A

Tetrahymena thermophila micronuclei contain four linker-associated proteins, alpha, beta, gamma, and delta. Synthetic oligonucleotides based on N-terminal protein sequences of beta and gamma were used to clone the micronuclear linker histone (MLH) gene. The MLH gene is single copy and is transcribed into a 2.4-kb message encoding all four linker-associated proteins. The message is translated into a polypeptide (Mic LH) that is processed at the sequence decreases RTK to give proteins whose amino acid sequences differ markedly from each other, from the sequence of macronuclear H1, and from sequences of typical H1s of other organisms. This represents the first example of multiple chromatin proteins derived from a single polyprotein. The delta protein consists largely of two high-mobility-group (HMG) boxes. An evolutionary analysis of HMG boxes indicates that the delta HMG boxes are similar to the HMG boxes of tsHMG, a protein that appears in elongating mouse spermatids when they condense and cease transcription, suggesting that delta could play a similar role in the micronucleus. The micronucleus divides mitotically, while the macronucleus divides amitotically. Surprisingly, macronuclear H1 but not Mic LH contains sequences resembling p34cdc2 kinase phosphorylation sites, while each of the Mic LH-derived proteins contains a typical protein kinase A phosphorylation site in its carboxy terminus.

Crystal structure and solution conformation of 1-N-acetyl-β-D-glucopyranosyl amine: A model for the glycopeptide linkage

Biopolymers ◽  
1982 ◽  
Vol 21 (10) ◽  
pp. 1971-1977 ◽  
Author(s):  
C. Allen Bush ◽  
Karl Blumberg ◽  
Joe N. Brown
Keyword(s):  
Crystal Structure ◽  
Solution Conformation

Distinctive Solution Conformation of Phosphatase Inhibitor CPI-17 Substituted with Aspartate at the Phosphorylation-site Threonine Residue

2003 ◽  
Vol 326 (5) ◽  
pp. 1539-1547 ◽  
Author(s):  
Shin-ya Ohki ◽  
Masumi Eto ◽  
Masato Shimizu ◽  
Rei Takada ◽  
David L. Brautigan ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Solution Conformation ◽  
Threonine Residue ◽  
Phosphatase Inhibitor

scholarly journals Characterization of Protein Kinase A and Protein Kinase C Phosphorylation of theN-Methyl-D-aspartate Receptor NR1 Subunit Using Phosphorylation Site-specific Antibodies

1997 ◽  
Vol 272 (8) ◽  
pp. 5157-5166 ◽  
Author(s):  
Whittemore G. Tingley ◽  
Michael D. Ehlers ◽  
Kimihiko Kameyama ◽  
Carol Doherty ◽  
Janine B. Ptak ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Phosphorylation Site ◽  
Site Specific ◽  
Specific Antibodies ◽  
Kinase C ◽  
Aspartate Receptor ◽  
Kinase A

scholarly journals Time-Dependent Activation of Phox2a by the Cyclic AMP Pathway Modulates Onset and Duration of p27Kip1 Transcription

2009 ◽  
Vol 29 (18) ◽  
pp. 4878-4890 ◽  
Author(s):  
Min Hwa Shin ◽  
Nirmala Mavila ◽  
Wen-Horng Wang ◽  
Sasha Vega Alvarez ◽  
Mark C. Hall ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Neuronal Differentiation ◽  
Transcriptional Activity ◽  
Phosphorylation Site ◽  
Time Dependent ◽  
Camp Signaling ◽  
Cell Cycle Exit ◽  
Camp Pathway ◽  
Cad Cells ◽  
Kinase A

ABSTRACT In noradrenergic progenitors, Phox2a mediates cell cycle exit and neuronal differentiation by inducing p27Kip1 transcription in response to activation of the cyclic AMP (cAMP) pathway. The mechanism of cAMP-mediated activation of Phox2a is unknown. We identified a cluster of phosphoserine-proline sites in Phox2a by mass spectrometry. Ser206 appeared to be the most prominent phosphorylation site. A phospho-Ser206 Phox2a antibody detected dephosphorylation of Phox2a that was dependent on activation of the cAMP pathway, which occurred prior to neuronal differentiation of noradrenergic CAD cells. Employing serine-to-alanine and serine-to-aspartic acid Phox2a substitution mutants expressed in inducible CAD cell lines, we demonstrated that the transcriptional activity of Phox2a is regulated by two sequential cAMP-dependent events: first, cAMP signaling promotes dephosphorylation of Phox2a in at least one site, Ser206, thereby allowing Phox2a to bind DNA and initiate p27Kip1 transcription; second, following dephosphorylation of the phosphoserine cluster (Ser202 and Ser208), Phox2a becomes phosphorylated by protein kinase A (PKA) on Ser153, which prevents association of Phox2a with DNA and terminates p27Kip1 transcription. This represents a novel mechanism by which the same stimulus, cAMP signaling, first activates Phox2a by dephosphorylation of Ser206 and then, after a built-in delay, inactivates Phox2a via PKA-dependent phosphorylation of Ser153, thereby modulating onset and duration of p27Kip1 transcription.

scholarly journals Completing the Partially Resolved Complex Crystal Structure of Aurora Kinase A-/-N-MYC by Molecular Modeling

2021 ◽  
Vol 120 (3) ◽  
pp. 19a-20a
Author(s):  
Pinar Altiner ◽  
Suleyman Selim Çınaroglu ◽  
Emel Timucin
Keyword(s):  
Crystal Structure ◽  
Molecular Modeling ◽  
Aurora Kinase ◽  
Aurora Kinase A ◽  
Complex Crystal Structure ◽  
Complex Crystal ◽  
Kinase A

Solution Conformation of the Antibody-Bound Tyrosine Phosphorylation Site of the Nicotinic Acetylcholine Receptor β-Subunit in Its Phosphorylated and Nonphosphorylated States†

Biochemistry ◽  
2003 ◽  
Vol 42 (24) ◽  
pp. 7371-7380 ◽  
Author(s):  
Angélique Phan-Chan-Du ◽  
Christine Hemmerlin ◽  
Dimitrios Krikorian ◽  
Maria Sakarellos-Daitsiotis ◽  
Vassilios Tsikaris ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Phosphorylation Site ◽  
Β Subunit ◽  
Solution Conformation ◽  
Nicotinic Acetylcholine ◽  
Tyrosine Phosphorylation Site

scholarly journals Endogenous cleavage of the Arg-379-Ala-380 bond in vitronectin results in a distinct conformational change which ‘buries’ Ser-378, its site of phosphorylation by protein kinase A

1991 ◽  
Vol 274 (2) ◽  
pp. 387-394 ◽  
Author(s):  
D Chain ◽  
B Korc-Grodzicki ◽  
T Kreizman ◽  
S Shaltiel
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Blood Platelets ◽  
Phosphorylation Site ◽  
Factor Xa ◽  
Heparin Binding ◽  
Recognition Sequence ◽  
The One ◽  
Chain Form ◽  
Kinase A

Activation of blood platelets by thrombin was previously shown to specifically release protein kinase A, which in human plasma singles out and phosphorylates one protein, identified as vitronectin. This protein is known to be involved in processes that follow platelet stimulation, specifically, in the binding of heparin (interfering with the heparin-mediated inhibition of thrombin and Factor Xa by antithrombin III), in the growth of endothelial cells and in fibrinolysis. This paper shows that phosphorylation of vitronectin by protein kinase A is stoichiometric (approx. 1 mol/mol), that it is targeted to one site (Ser-378) at the C-terminal edge of the heparin-binding domain, and that it distinguishes between the two physiologically occurring forms of vitronectin: the one-chain (75 kDa) form, and the nicked two-chain (65 + 10 kDa) form, held together by an interchain disulphide bridge. Protein kinase A phosphorylates the one-chain form but not the two-chain form, although Ser-378 and the complete recognition sequence of the kinase are still present in the clipped 65 kDa chain. Cleavage of the Arg-379-Ala-380 bond results therefore in a conformationally distinct form of vitronectin in which Ser-378 is ‘buried’. This is demonstrated by our finding that Ser-378 is present in the 65 kDa chain of clipped vitronectin but inaccessible to phosphorylation at physiological pH. Upon binding heparin, the phosphorylation site becomes exposed and able to undergo a stoichiometric phosphorylation at physiological pH.

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