scholarly journals Protein Kinase Activity and Identification of a Toxic Effector Domain of the Target of Rapamycin TOR Proteins in Yeast

1999 ◽  
Vol 10 (8) ◽  
pp. 2531-2546 ◽  
Author(s):  
Clara M. Alarcon ◽  
Joseph Heitman ◽  
Maria E. Cardenas
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Tor Signaling ◽  
Site Mutation ◽  
Effector Domain ◽  
Kinase Cascade

In complex with FKBP12, the immunosuppressant rapamycin binds to and inhibits the yeast TOR1 and TOR2 proteins and the mammalian homologue mTOR/FRAP/RAFT1. The TOR proteins promote cell cycle progression in yeast and human cells by regulating translation and polarization of the actin cytoskeleton. A C-terminal domain of the TOR proteins shares identity with protein and lipid kinases, but only one substrate (PHAS-I), and no regulators of the TOR-signaling cascade have been identified. We report here that yeast TOR1 has an intrinsic protein kinase activity capable of phosphorylating PHAS-1, and this activity is abolished by an active site mutation and inhibited by FKBP12-rapamycin or wortmannin. We find that an intact TOR1 kinase domain is essential for TOR1 functions in yeast. Overexpression of a TOR1 kinase-inactive mutant, or of a central region of the TOR proteins distinct from the FRB and kinase domains, was toxic in yeast, and overexpression of wild-type TOR1 suppressed this toxic effect. Expression of the TOR-toxic domain leads to a G1 cell cycle arrest, consistent with an inhibition of TOR function in translation. Overexpression of the PLC1gene, which encodes the yeast phospholipase C homologue, suppressed growth inhibition by the TOR-toxic domains. In conclusion, our findings identify a toxic effector domain of the TOR proteins that may interact with substrates or regulators of the TOR kinase cascade and that shares sequence identity with other PIK family members, including ATR, Rad3, Mei-41, and ATM.

HsRec2/Rad51L1, a Protein Influencing Cell Cycle Progression, Has Protein Kinase Activity

2000 ◽  
Vol 254 (1) ◽  
pp. 33-44 ◽  
Author(s):  
Pamela A. Havre ◽  
Michael Rice ◽  
Ronald Ramos ◽  
Eric B. Kmiec
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Cycle Progression

scholarly journals The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle.

1991 ◽  
Vol 11 (8) ◽  
pp. 4045-4052 ◽  
Author(s):  
S Garrett ◽  
M M Menold ◽  
J R Broach
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
S Phase ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Dependent Protein

Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase). That is, loss of the YAK1 protein specifically compensates for loss of the A kinase. Here, we show that the protein encoded by YAK1 has protein kinase activity. Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase. Induction is not observed by arrest at stages later in the cell cycle. Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity. Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity. These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

scholarly journals Tyr198 is the Essential Autophosphorylation Site for STK16 Localization and Kinase Activity

2019 ◽  
Vol 20 (19) ◽  
pp. 4852 ◽  
Author(s):  
Junjun Wang ◽  
Juanjuan Liu ◽  
Xinmiao Ji ◽  
Xin Zhang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Serine Threonine Kinase ◽  
Cycle Progression ◽  
Threonine Kinase ◽  
Cellular Processes ◽  
Activation Segment ◽  
And Function

STK16, reported as a Golgi localized serine/threonine kinase, has been shown to participate in multiple cellular processes, including the TGF-β signaling pathway, TGN protein secretion and sorting, as well as cell cycle and Golgi assembly regulation. However, the mechanisms of the regulation of its kinase activity remain underexplored. It was known that STK16 is autophosphorylated at Thr185, Ser197, and Tyr198 of the activation segment in its kinase domain. We found that STK16 localizes to the cell membrane and the Golgi throughout the cell cycle, but mutations in the auto-phosphorylation sites not only alter its subcellular localization but also affect its kinase activity. In particular, the Tyr198 mutation alone significantly reduced the kinase activity of STK16, abolished its Golgi and membrane localization, and affected the cell cycle progression. This study demonstrates that a single site autophosphorylation of STK16 could affect its localization and function, which provides insights into the molecular regulatory mechanism of STK16’s kinase activity.

scholarly journals Mapping of a self-interaction domain of the cytomegalovirus protein kinase pUL97

2007 ◽  
Vol 88 (2) ◽  
pp. 395-404 ◽  
Author(s):  
Vera Schregel ◽  
Sabrina Auerochs ◽  
Ramona Jochmann ◽  
Katja Maurer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Interaction Domain ◽  
Amino Acid Region ◽  
Substrate Phosphorylation ◽  
Regulatory Functions ◽  
Acid Region

The human cytomegalovirus-encoded protein kinase pUL97 is a determinant of efficient virus replication and fulfils several regulatory functions. In particular, pUL97 interacts with and phosphorylates viral and cellular proteins. Substrate phosphorylation has regulatory consequences on viral replicative stages such as DNA synthesis, transcription and nuclear capsid egress. pUL97, in accordance with related herpesviral protein kinases, possesses strong autophosphorylation activity. Here, we demonstrate that pUL97 shows a pronounced potential to self-interact. Self-interaction of pUL97 is not dependent on its kinase activity, as seen with a catalytically inactive point mutant. The property of self-interaction maps to the amino acid region 231–280 which is separable from the postulated kinase domain. The detection of high-molecular-mass complexes of pUL97 suggests the formation of dimers and oligomers. Importantly, the analysis of pUL97 mutants by in vitro kinase assays demonstrated a correlation between self-interaction and protein kinase activity, i.e. all mutants lacking the ability to self-interact were negative or reduced in their kinase activity. Thus, our findings provide novel insights into the pUL97 structure–activity relationship suggesting an importance of self-interaction for pUL97 functionality.

scholarly journals Regulation of cyclin D1 expression and cell cycle progression by mitogen-activated protein kinase cascade

1999 ◽  
Vol 56 (4) ◽  
pp. 1258-1261 ◽  
Author(s):  
Yoshio Terada ◽  
Seiji Inoshita ◽  
Osamu Nakashima ◽  
Michio Kuwahara ◽  
Sei Sasaki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Mitogen Activated Protein Kinase ◽  
Cycle Progression ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

1999 ◽  
Vol 19 (5) ◽  
pp. 3877-3884 ◽  
Author(s):  
Akihiro Kurimasa ◽  
Satoshi Kumano ◽  
Nikolai V. Boubnov ◽  
Michael D. Story ◽  
Chang-Shung Tung ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Strand Breaks ◽  
Human Dna ◽  
Dna Dsbs ◽  
Dependent Protein ◽  
Dna Strand

ABSTRACT The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos

Development ◽  
1991 ◽  
Vol 113 (3) ◽  
pp. 789-795 ◽  
Author(s):  
T. Choi ◽  
F. Aoki ◽  
M. Mori ◽  
M. Yamashita ◽  
Y. Nagahama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Polar Body ◽  
Mitotic Cell ◽  
Histone H1 ◽  
Protein Kinase Activity ◽  
Mitotic Cell Cycle ◽  
Mouse Oocytes ◽  
First Polar Body

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.

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