Mouse and human La proteins differ in kinase substrate activity and activation mechanism for tRNA processing.
The La protein interacts with a variety of small RNAs as well as certain growth-associated mRNAs such as Mdm2 mRNA. Human La (hLa) phosphoprotein is so highly conserved that it can replace the tRNA processing function of the fission yeast La protein in vivo. We used this system, which is based on tRNA-mediated suppression (TMS) of ade6-704 in S. pombe, to compare the activities of mouse and human La proteins. Prior studies indicate that hLa is activated by phosphorylation of serine-366 by protein kinase CK2, neutralizing a negative effect of a short basic motif (SBM). First, we report the sequence mapping of the UGA stop codon that requires suppressor tRNA for TMS, to an unexpected site in S. pombe ade6-704. Next, we show that, unlike hLa, native mLa is unexpectedly inactive for TMS, although its intrinsic activity is revealed by deletion of its SBM. We then show that mLa is not phosphorylated by CK2, accounting for the mechanistic difference between mLa and hLa. We found a PKA/PKG target sequence in mLa (S199) that is not present in hLa, and show that PKA/PKG efficiently phosphorylates mLa S199 in vitro. A noteworthy conclusion that comes from this work is that this fission yeast system can be used to gain insight into differences in control mechanisms used by La proteins of different mammalian species. Finally, RNA binding assays indicate that while mutation of mLa S199 has little effect on pre-tRNA binding, it substantially decreases binding to a probe derived from Mdm2 mRNA. In closing, we note that species-specific signaling through La may be relevant to the La-dependent Mdm2 pathways of p53 metabolism and cancer progression in mice and humans.
Park
Jung-Min
JM
Intramural Research Program, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2426, USA.
Intine
Robert V
RV
Maraia
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000097585:SS-B Antigen
D001324:Autoantigens; D011494:Protein Kinases; D051736:Proto-Oncogene Proteins c-mdm2; D012343:RNA, Transfer; D012261:Ribonucleoproteins; D000097585:SS-B Antigen