The "atypical" protein kinase, SsoPK5, an archaeal member of the piD261/Bud32 subfamily

Open reading frame (ORF) sso0433 from the archaeon Sulfolobus solfataricus encodes a protein kinase, SsoPK5 that exhibits 33% sequence identity to p53 related protein kinase (PRPK) from Homo sapiens and 26% sequence identity to piD261/Bud32 from Saccharomyces cerevisiae. Given this high degree of similarity, the objectives of this thesis were to (a) clone and purify recombinant SsoPK5, (b) examine its commonalities and differences with its eukaryotic homologues, and (c) determine if it was regulated by nucleotides or related compounds. Substantial progress was achieved on each objective. After successful cloning of ORF sso0433 and purification of its protein product, SsoPK5, it was determined that SsoPK5 was cold labile and incubation at 4ºC for an extended period of time rendered SsoPK5 incapable of phosphotransferase activity. When stored at room temperature, SsoPK5 was capable of transferring the γ-phosphate from ATP to casein, reduced carboxyamidomethylated and maleylated (RCM) lysozyme,and p53. SsoPK5 phosphotransferase activity required a divalent metal cofactor; like pid261/Bud32, SsoPK5 preferred Mn²⁺ over the more commonly preferred Mg²⁺. SsoPK5 was shown to phosphorylate itself on threonine and serine residues; one of the specific amino acid residues modified is threonine-151. Recombinant SsoPK5 is activated by ADP-ribose and 5′-AMP. Activation was observed when SsoPK5 was stabilized by ATP or a nonhydrolytic analogue, such as β,γ- methylene adenosine 5′-triphosphate (AMP-PCP). Activation was not a result of phosphoryl transfer nor hydrolytic breakdown of ATP or 5′-AMP. This was deduced by the lack of ³²P radioactivity incorporated into SsoPK5 during pre-incubation with [γ-³²P] ATP for 60 min at 65ºC, and activation by adenosine 5′-O-thiomonophosphate (AMPS), a hydrolysis-resistant analog of AMP. These results may indicate that ADP-ribose acts as a pseudochaperone for SsoPK5 thereby facilitating maximal activity.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry

Department dc:contributor.department

Biochemistry

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2009

Author dc:creator

• Haile, January Dendi

Chair dc:contributor.committeechair

• Kennelly, Peter J.

Committee members dc:contributor.committeemember

• Mahaney, James M.
• Dolan, Erin L.
• Tu, Zhijian Jake

• protein phosphorylation
• piD261/Bud32
• ADP-ribose
• Archaea

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Dc Identifier Other

etd-08132009-165600

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/39127