scholarly journals The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2053
Author(s):  
Dalma Ménesi ◽  
Éva Klement ◽  
Györgyi Ferenc ◽  
Attila Fehér
Keyword(s):  
Protein Kinases ◽  
Phosphorylation Site ◽  
Interaction Strength ◽  
Yeast Cells ◽  
Phosphorylation Sites ◽  
Mutant Proteins ◽  
In Vivo Test ◽  
Calcium Dependent

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

scholarly journals Regulation of p53 Function and Stability by Phosphorylation

1999 ◽  
Vol 19 (3) ◽  
pp. 1751-1758 ◽  
Author(s):  
Margaret Ashcroft ◽  
Michael H. G. Kubbutat ◽  
Karen H. Vousden
Keyword(s):  
Dna Damage ◽  
Protein Kinases ◽  
Phosphorylation Site ◽  
Tumor Suppressor Protein ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Mutant Proteins ◽  
Encoding Gene ◽  
P53 Tumor Suppressor Protein

ABSTRACT The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

scholarly journals Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

1996 ◽  
Vol 7 (10) ◽  
pp. 1573-1586 ◽  
Author(s):  
K L Gould ◽  
A Feoktistova
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Phosphorylation Site ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Mutant Proteins ◽  
Regulatory Phosphorylation ◽  
Binding Loop

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

scholarly journals Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae

2003 ◽  
Vol 23 (10) ◽  
pp. 3405-3416 ◽  
Author(s):  
Julia M. Sidorova ◽  
Linda L. Breeden
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Phosphorylation Site ◽  
Site Preference ◽  
Phosphorylation Sites ◽  
Checkpoint Kinase ◽  
Checkpoint Signaling ◽  
And Function

ABSTRACT Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes. When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division. Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity. A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect. Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G1-specific transcriptional activator, is a substrate of Rad53 in the G1/S DNA damage checkpoint. In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo. Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G1-to-S transition after DNA damage. We derive a consensus for Rad53 site preference at positions −2 and +2 (−2/+2) and identify its potential substrates in the yeast proteome. Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.

scholarly journals Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Masayasu Okada ◽  
Yosuke Kawagoe ◽  
Yuta Sato ◽  
Motohiro Nozumi ◽  
Yuya Ishikawa ◽  
...  
Keyword(s):  
Molecular Marker ◽  
Phosphorylation Site ◽  
Axon Growth ◽  
Growth Cones ◽  
Specific Protein ◽  
Phosphorylation Sites ◽  
Quarter Century ◽  
Wide Range

AbstractGAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

scholarly journals The relative roles of specific N- and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells

1996 ◽  
Vol 109 (4) ◽  
pp. 817-826 ◽  
Author(s):  
Y.H. Chou ◽  
P. Opal ◽  
R.A. Quinlan ◽  
R.D. Goldman
Keyword(s):  
Protein Kinase ◽  
Phosphorylation Site ◽  
Supramolecular Organization ◽  
Phosphorylation Sites ◽  
Bhk Cells ◽  
Terminal Domain ◽  
Kinase Phosphorylation ◽  
Mitotic Cells

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.

scholarly journals Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

2004 ◽  
Vol 381 (2) ◽  
pp. 471-481 ◽  
Author(s):  
Mark E. GRAHAM ◽  
Patricia RUMA-HAYNES ◽  
Amanda G. CAPES-DAVIS ◽  
Joanne M. DUNN ◽  
Timothy C. TAN ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Site Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Single Mutation ◽  
Cyclin Dependent Kinase ◽  
Major Site ◽  
Multisite Phosphorylation ◽  
Cyclin Dependent Kinase 5

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.

scholarly journals T. cruzi DNA polymerase beta (Tcpolβ) is phosphorylated in vitro by CK1, CK2 and TcAUK1 leading to the potentiation of its DNA synthesis activity

2021 ◽  
Vol 15 (7) ◽  
pp. e0009588
Author(s):  
Edio Maldonado ◽  
Diego A. Rojas ◽  
Fabiola Urbina ◽  
Aldo Solari
Keyword(s):  
Protein Kinases ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Polymerase Activity ◽  
Casein Kinase ◽  
Phosphorylation Sites ◽  
Casein Kinase 1 ◽  
Synthesis Activity

The unicellular protozoan Trypanosoma cruzi is the causing agent of Chagas disease which affects several millions of people around the world. The components of the cell signaling pathways in this parasite have not been well studied yet, although its genome can encode several components able to transduce the signals, such as protein kinases and phosphatases. In a previous work we have found that DNA polymerase β (Tcpolβ) can be phosphorylated in vivo and this modification activates the synthesis activity of the enzyme. Tcpolβ is kinetoplast-located and is a key enzyme in the DNA base excision repair (BER) system. The polypeptide possesses several consensus phosphorylation sites for several protein kinases, however, a direct phosphorylation of those sites by specific kinases has not been reported yet. Tcpolβ has consensus phosphorylation sites for casein kinase 1 (CK1), casein kinase 2 (CK2) and aurora kinase (AUK). Genes encoding orthologues of those kinases exist in T. cruzi and we were able to identify the genes and to express them to investigate whether or no Tcpolβ could be a substrate for in vitro phosphorylation by those kinases. Both CK1 and TcAUK1 have auto-phosphorylation activities and they are able to phosphorylate Tcpolβ. CK2 cannot perform auto-phosphorylation of its subunits, however, it was able to phosphorylate Tcpolβ. Pharmacological inhibitors used to inhibit the homologous mammalian kinases can also inhibit the activity of T. cruzi kinases, although, at higher concentrations. The phosphorylation events carried out by those kinases can potentiate the DNA polymerase activity of Tcpolβ and it is discussed the role of the phosphorylation on the DNA polymerase and lyase activities of Tcpolβ. Taken altogether, indicates that CK1, CK2 and TcAUK1 can play an in vivo role regulating the function of Tcpolβ.

scholarly journals CK2 Controls Multiple Protein Kinases by Phosphorylating a Kinase-Targeting Molecular Chaperone, Cdc37

2004 ◽  
Vol 24 (9) ◽  
pp. 4065-4074 ◽  
Author(s):  
Yoshihiko Miyata ◽  
Eisuke Nishida
Keyword(s):  
Protein Kinases ◽  
Molecular Chaperone ◽  
Regulatory Mechanism ◽  
Phosphorylation Site ◽  
Binding Activity ◽  
Serine Residue ◽  
Cellular Functions ◽  
Multiple Protein

ABSTRACT Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.

scholarly journals Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death inSaccharomyces cerevisiae

2006 ◽  
Vol 75 (1) ◽  
pp. 417-428 ◽  
Author(s):  
Xiao-Ping Li ◽  
Marianne Baricevic ◽  
Hemalatha Saidasan ◽  
Nilgun E. Tumer
Keyword(s):  
Cell Death ◽  
Large Scale ◽  
Chromatin Condensation ◽  
Yeast Cells ◽  
Plant Toxin ◽  
Mutant Proteins ◽  
Catalytically Active ◽  
A Chain

ABSTRACT The plant toxin ricin is one of the most potent and lethal substances known. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved α-sarcin/ricin loop in the large rRNA. Very little is known about how ricin interacts with ribosomes and the molecular mechanism by which it kills cells. To gain insight to the mechanism of ricin-induced cell death, we set up yeast (Saccharomyces cerevisiae) as a simple and genetically tractable system to isolate mutants defective in cytotoxicity. Ribosomes were depurinated in yeast cells expressing the precursor form of the A chain of ricin (pre-RTA), and these cells displayed apoptotic markers such as nuclear fragmentation, chromatin condensation, and accumulation of reactive oxygen species. We conducted a large-scale mutagenesis of pre-RTA and isolated a panel of nontoxic RTA mutants based on their inability to kill yeast cells. Several nontoxic RTA mutants depurinated ribosomes and inhibited translation to the same extent as wild-type RTA in vivo. The mutant proteins isolated from yeast depurinated ribosomes in vitro, indicating that they were catalytically active. However, cells expressing these mutants did not display hallmarks of apoptosis. These results provide the first evidence that the ability to depurinate ribosomes and inhibit translation does not always correlate with ricin-mediated cell death, indicating that ribosome depurination and translation inhibition do not account entirely for the cytotoxicity of ricin.

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