Translation of p15.5INK4B, an N-terminally extended and fully active form of p15INK4B, is initiated from an upstream GUG codon

Abstract

The expression of the cyclin-dependent kinase inhibitor p15 INK4B in normal cells after induction with TGF-b1, or following overexpression from an adenovirus-encoded cDNA, appears on an SDS-polyacrylamide gel as a doublet. Here, the underlying mechanism behind the synthesis of the two species has been studied. By expressing cDNAs truncated at their 5' end, we found that the synthesis of the more slowly migrating form, called p15.5 INK4B , is dependent on a sequence upstream of the ®rst AUG codon thought to initiate translation of p15 INK4B . Two potential, in frame, alternative upstream initiation codons, ACG and GUG, were individually changed to GCA encoding alanine. Analysis by in vitro translation, or immunoblotting of lysates from transfected 293 cells, showed that translation of p15.5 INK4B is initiated at the GUG located 13 codons upstream of the ®rst AUG. When this AUG was mutated, p15 INK4B was no longer made. Instead, a shorter form, initiated at an in frame AUG located seven codons downstream, was synthesized. Finally, when both these AUGs were mutated, only p15.5 INK4B was generated. Both p15 INK4B and p15.5 INK4B bound to CDK4 and CDK6, inhibited DNA synthesis, and caused replicative senescence of a human glioma cell line. We thus conclude that p15 INK4B and p15.5 INK4B , encoded by the CDKN2B gene, are functionally indistinguishable as based on these assays.

Regulation of translation via mRNA structure in prokaryotes and eukaryotes

“…The weakness explains why non-AUG codons are usually used only as supplementary initiation sites; i.e. ribosomes initiate at an upstream non-AUG codon in addition to initiating at the first AUG (Carroll and Derse, 1993;Chang and Wang, 2004;Fütterer et al, 1997;Fuxe et al, 2000;Portis et al, 1994). The non-AUG initiated protein serves a useful function in those examples; but in some other cases, the N-terminally extended form of the protein has no biological relevance (Miles et al, 2003).…”

Anti-Epidermal Growth Factor Receptor Monoclonal Antibody 225 Upregulates p27^KIP1 and p15^INK4B and Induces G1 Arrest in Oral Squamous Carcinoma Cell Lines

“…On SDS-polyacrylamide gels, we consistently observed that p15 INK4B migrated as a doublet. These two species, called p15.5 INK4B and p15 INK4B , were analyzed to determine the molecular and possible functional differences, also reported by Fuxe et al [20]. They concluded that there were no functional differences in the capacity of the two forms.…”

Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

“…This may be explained by the fact that our initial mutation of the primary AUG to GUG did not affect levels of luciferase activity, indicating that this mutation does not completely abolish the potency of the translation start codon. In fact GUG has been shown in other systems to function as an alternative start codon (19). Further mutation of the start site and the flanking nucleotides attenuated ribosome binding, but it did not completely abolish it.…”