Kinase Family PIM

Kinase Classification: Group CAMK: Family PIM

Individual Notes

Unusual Start of PIM1 protein in mammals

Pim1 has an unusual start to the protein, in mouse and probably other species. While all species have a conserved start site ATG codon with a protein start (in human) of MLLSKINSLA, a careful study by Saris et al [1] showed in mouse that the 34kD product of this ORF was accompanied by a 44kd protein, which they showed to initiate upstream in the same exon, at a CTG (Leucine) codon, extending the protein by 84 AA (start = MGPAAPLALP). Mutation or deletion of the CTG abolished the longer isoform.

The human gene also has a long 5' UTR in the same exon as the ORF start, has a CTG codon at the same position as in mouse, and the upstream extension of the gene is moderately similar to that of mouse. The Saris paper cites unpublished data that only one human protein isoform was seen in the the tissue they tested, but a longer human form may also be expressed under particular conditions.

The long 5' UTR of Pim1 (including the putative upstream protein extension) is highly conserved in DNA, forms a predicted RNA secondary structure (free energy of -153 kCal/mol) and appears to control translation of the protein [2]. Deletion of the region in human (including the predicted upstream extension above the ATG) resulted in ~10-fold increase in in vitro translation (and a modest in vivo effect), and this effect was modulated by changing eIF4E levels. A subsequent paper [3] identified human Pim1 in a screen for transcripts with cap-independent translation and showed that the 5' UTR contains an internal ribosome entry sites (IRES), though a later publication claims this as a cryptic promoter [4]. This apparent function of the UTR complicates our ability to predict whether an extension of the ORF into this region is conserved for protein-coding regions or otherwise. Conversely, the IRES may assist with the non-canonical translation start.

There is strong conservation of the UTR and immediately upstream genomic region beyond this extended start, indicating that the extended ORF is not the only functional element in this region. An ORF extends upstream to an aligning CTG in rhesus, rat, and dog. By contrast, there is a stop just two AA upstream from the ATG start in ESTs and genomic sequence from Fugu and Stickleback, and the upstream UTRs in zebra finch, Xenopus tropicalis and Medaka have little or no protein sequence similarity to those of the mammals, suggesting that this longer ORF is restricted to mammals. Many other genomes (horse, cow, marmoset, guinea pig, chicken) have gaps or poor sequence regions just upstream of the ATG, possibly due to difficulty in cloning and maintenance of sequences with high secondary structure.

The shorter PIM1 protein (ATG start) aligns well to the start of the PIM3 protein in human and mouse.

References

1. Saris CJ, Domen J, and Berns A. The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG. EMBO J. 1991 Mar;10(3):655-64. DOI:10.1002/j.1460-2075.1991.tb07994.x |
2. Hoover DS, Wingett DG, Zhang J, Reeves R, and Magnuson NS. Pim-1 protein expression is regulated by its 5'-untranslated region and translation initiation factor elF-4E. Cell Growth Differ. 1997 Dec;8(12):1371-80.
3. Johannes G, Carter MS, Eisen MB, Brown PO, and Sarnow P. Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. Proc Natl Acad Sci U S A. 1999 Nov 9;96(23):13118-23. DOI:10.1073/pnas.96.23.13118 |
4. Wang Z, Weaver M, and Magnuson NS. Cryptic promoter activity in the DNA sequence corresponding to the pim-1 5'-UTR. Nucleic Acids Res. 2005;33(7):2248-58. DOI:10.1093/nar/gki523 |