Kinase Family Bud32

Kinase Classification: Group PKL: Family Bud32

Bud32 is an ancient protein kinase, found across eukaryotes and archaea, involved in tRNA modification and telomere regulation. The human form appears to be a p53 regulator.

Evolution

Bud32 is found in all eukaryotes and archaea examined, usually as a single copy. The gene is called Bud32 in yeast, and PRPK/TP53RK in human. It is part of the conserved KEOPS complex that regulates tRNA modification, telomere length, and may activate p53 and glutaredoxins in humans.

Domain Structure

Most Bud32s consist of a kinase domain and a short N-terminal extension. Some archaeal members are fused to Kae1, another member of the KEOPS complex (see below). The Bud32 kinase domain has a PKL fold, but is distinct from the ePK superfamily to which most eukaryotic protein kinases belong. It is most similar to KdoK (a lipopolysaccharide kinase found in some gammaproteobacteria) and to Rio, the other kinase family found in all eukaryotes and archaea [1].

Functions

Bud32 is a member of the KEOPS complex, whose other members are Kae1, Cgi121, Gon7, and Pcc1 (in yeast; human proteins are OSGEP, TPRKB, GON7 and LAGE3, respectively). This complex has several roles, including telomere maintenance [2], homologous recombination and DNA damage repair [3], transcriptional regulation, and bud site selection in yeast, but it's most conserved and studied role is in modification of tRNAs that decode codons that start with A, by adding t6A (N6-threonylcarbamoyladenosine) to residue 37 of these tRNAs, an essential modification in all organisms (bacteria and mitochondria have Kae1 homologs but not the full complex). The catalytic activity of Bud32 is required for the tRNA modification, but it is reported that Bud32 acts as an ATPase, releasing free phosphate, in this context [4]

Bud32 has a diminished C-lobe, losing some of the substrate-interaction regions. It is known to autophosphorylate [4], and catalytic residues are required for viability.

Human PRPK has been shown to bind, phosphorylate, and activate p53 [5]. PRPK can phosphorylate p53 on Ser-15, both in vitro and in cell transfections, though this has also been suggested to be an artefact [6]. Ser-15 is also phosphorylated by several other kinases, including the DNA damage kinases ATM and ATR, causing stabilization through recruitment of p300. PRPK only functioned after activation by cell lysate, later traced to phosphorylation on Ser-250 by Akt [7], though it is not known if this is physiologically relevant. TPRKB/CGI-121 blocks binding of PRPK to p53, suggesting that the PRPK that binds p53 is not within the KEOPS complex [8].

PRPK has been reported to interact with the small GTPase, RAB35 (ray, Rab1C), causing it's proteosomal degradation in neurons, a function that is blocked by MAP1B microtubule-associated protein [9], while RAB35 was also shown to sequester PRPK in the cytoplasm, preventing it from activating p53 [10]

Yeast Bud32 phosphorylates and activates the Grx4 glutaredoxin on Ser-134 [11]. This is enhanced by posphorylation on Ser-258 by Sch9, a yeast Akt homolog, which did not impact Bud32 kinase activity, but increased Grx4 binding. Ser-258 is the equivalent of human PRPK Ser-250 (also an Akt site, see above), and both are in a conserved Akt-consensus site RGRK[RK][TS], though this motif is also conserved in organisms that do not have S/T at this site. PRPK was also shown to bind human glutaredoxin GLRX3 in a high-throughput cofractionation study [12]

Yeast Bud32 mutants show random budding patterns, as do other KEOPS mutations. The KEOPS complex is required for proper localization of the budding protein Bud9 [13], though the mechanism is not known.

The Drosophila KEOPS complex has been implicated as a regulator of the TOR kinase pathway [14]. Loss of KEOPS members leads to activation of the unfolded protein response (UPR), possibly due to translation errors caused by lack of t6A [15]. A link between TOR signaling and KEOPS has also been found in yeast [16].

References

1. Kannan N, Taylor SS, Zhai Y, Venter JC, and Manning G. Structural and functional diversity of the microbial kinome. PLoS Biol. 2007 Mar;5(3):e17. DOI:10.1371/journal.pbio.0050017 |
2. Downey M, Houlsworth R, Maringele L, Rollie A, Brehme M, Galicia S, Guillard S, Partington M, Zubko MK, Krogan NJ, Emili A, Greenblatt JF, Harrington L, Lydall D, and Durocher D. A genome-wide screen identifies the evolutionarily conserved KEOPS complex as a telomere regulator. Cell. 2006 Mar 24;124(6):1155-68. DOI:10.1016/j.cell.2005.12.044 |
3. He MH, Liu JC, Lu YS, Wu ZJ, Liu YY, Wu Z, Peng J, and Zhou JQ. KEOPS complex promotes homologous recombination via DNA resection. Nucleic Acids Res. 2019 Jun 20;47(11):5684-5697. DOI:10.1093/nar/gkz228 |
4. Perrochia L, Guetta D, Hecker A, Forterre P, and Basta T. Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification. Nucleic Acids Res. 2013 Nov;41(20):9484-99. DOI:10.1093/nar/gkt720 |
5. Abe Y, Matsumoto S, Wei S, Nezu K, Miyoshi A, Kito K, Ueda N, Shigemoto K, Hitsumoto Y, Nikawa J, and Enomoto Y. Cloning and characterization of a p53-related protein kinase expressed in interleukin-2-activated cytotoxic T-cells, epithelial tumor cell lines, and the testes. J Biol Chem. 2001 Nov 23;276(47):44003-11. DOI:10.1074/jbc.M105669200 |
6. Beenstock J and Sicheri F. The structural and functional workings of KEOPS. Nucleic Acids Res. 2021 Nov 8;49(19):10818-10834. DOI:10.1093/nar/gkab865 |
7. Facchin S, Ruzzene M, Peggion C, Sartori G, Carignani G, Marin O, Brustolon F, Lopreiato R, and Pinna LA. Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB. Cell Mol Life Sci. 2007 Oct;64(19-20):2680-9. DOI:10.1007/s00018-007-7179-7 |
8. Miyoshi A, Kito K, Aramoto T, Abe Y, Kobayashi N, and Ueda N. Identification of CGI-121, a novel PRPK (p53-related protein kinase)-binding protein. Biochem Biophys Res Commun. 2003 Apr 4;303(2):399-405. DOI:10.1016/s0006-291x(03)00333-4 |
9. Villarroel-Campos D, Henríquez DR, Bodaleo FJ, Oguchi ME, Bronfman FC, Fukuda M, and Gonzalez-Billault C. Rab35 Functions in Axon Elongation Are Regulated by P53-Related Protein Kinase in a Mechanism That Involves Rab35 Protein Degradation and the Microtubule-Associated Protein 1B. J Neurosci. 2016 Jul 6;36(27):7298-313. DOI:10.1523/JNEUROSCI.4064-15.2016 |
10. Abe Y, Takeuchi T, Imai Y, Murase R, Kamei Y, Fujibuchi T, Matsumoto S, Ueda N, Ogasawara M, Shigemoto K, and Kito K. A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK. Biochem Biophys Res Commun. 2006 May 26;344(1):377-85. DOI:10.1016/j.bbrc.2006.03.071 |
11. Facchin S, Lopreiato R, Stocchetto S, Arrigoni G, Cesaro L, Marin O, Carignani G, and Pinna LA. Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life. Biochem J. 2002 Jun 1;364(Pt 2):457-63. DOI:10.1042/BJ20011376 |
12. Wan C, Borgeson B, Phanse S, Tu F, Drew K, Clark G, Xiong X, Kagan O, Kwan J, Bezginov A, Chessman K, Pal S, Cromar G, Papoulas O, Ni Z, Boutz DR, Stoilova S, Havugimana PC, Guo X, Malty RH, Sarov M, Greenblatt J, Babu M, Derry WB, Tillier ER, Wallingford JB, Parkinson J, Marcotte EM, and Emili A. Panorama of ancient metazoan macromolecular complexes. Nature. 2015 Sep 17;525(7569):339-44. DOI:10.1038/nature14877 |
13. Kato Y, Kawasaki H, Ohyama Y, Morishita T, Iwasaki H, Kokubo T, and Hirano H. Cell polarity in Saccharomyces cerevisiae depends on proper localization of the Bud9 landmark protein by the EKC/KEOPS complex. Genetics. 2011 Aug;188(4):871-82. DOI:10.1534/genetics.111.128231 |
14. Ibar C, Cataldo VF, Vásquez-Doorman C, Olguín P, and Glavic A. Drosophila p53-related protein kinase is required for PI3K/TOR pathway-dependent growth. Development. 2013 Mar;140(6):1282-91. DOI:10.1242/dev.086918 |
15. Rojas-Benítez D, Ibar C, and Glavic Á. The Drosophila EKC/KEOPS complex: roles in protein synthesis homeostasis and animal growth. Fly (Austin). 2013 Jul-Sep;7(3):168-72. DOI:10.4161/fly.25227 |
16. Kessi-Pérez EI, Salinas F, González A, Su Y, Guillamón JM, Hall MN, Larrondo LF, and Martínez C. KAE1 Allelic Variants Affect TORC1 Activation and Fermentation Kinetics in Saccharomyces cerevisiae. Front Microbiol. 2019;10:1686. DOI:10.3389/fmicb.2019.01686 |