Type of Document Dissertation Author Haile, January Dendi URN etd-08132009-165600 Title The “atypical” protein kinase, SsoPK5, an archaeal member of the piD261/Bud32 subfamily Degree PhD Department Biochemistry Advisory Committee
Advisor Name Title Kennelly, Peter J. Committee Chair Dolan, Erin L. Committee Member Mahaney, James M. Committee Member Tu, Zhijian Jake Committee Member Keywords
- protein phosphorylation
- piD261/Bud32
- ADP-ribose
- Archaea
Date of Defense 2009-08-12 Availability restricted Abstract Open reading frame (ORF) sso0433 from the archaeon Sulfolobus solfataricusencodes a protein kinase, SsoPK5 that exhibits 33% sequence ident ity 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.
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