PTEN is an evolutionarily conserved protein and

PTEN is an evolutionarily conserved protein and has been considered to be genetically unique without other isoforms. In this study, we identified an alternate translation initiation at a CUG site in the 5′ untranslated region (5′ UTR) of PTEN mRNA. This CUG start Glucose Uptake Fluorometric Assay Kit generates a larger form of PTEN with an elongated N-terminal region comprising an additional 173 (Homo sapiens) or 169 (Mus musculus) amino acids. We used multiple approaches to demonstrate the existence of this new form of PTEN, which we have designated PTENα. We show that eIF2A-dependent CUG initiation is involved in PTENα synthesis and that a CUG-centered palindromic sequence is required for this process. PTENα is involved in the eukaryotic electron transport process through induction of cytochrome c oxidase activity in mitochondria, and disruption of PTENα impairs mitochondrial bioenergetics. These results establish that a PTEN isoform of greater length and additional functions is produced by an alternative CUG translation initiation. Identification of PTENα suggests reinterpretation of the importance of PTEN in multiple fundamental cellular activities is warranted.
Results
Discussion In eukaryotes, protein translation of mRNA is typically initiated at AUG codons, and the efficiency of initiation depends on the nucleotide context in which the initiator codon is embedded (Kozak, 1999). There is growing evidence, including the findings in this study, that shows translation initiation also occurs at non-AUG codons (Gerashchenko et al., 2010, Hann et al., 1988, Malarkannan et al., 1999, Németh et al., 2007), which enhances genome coding capacity and protein diversity. While CUG appears to be the most common non-AUG initiation codon (Peabody, 1989, Wegrzyn et al., 2008), the mechanism underlying CUG initiation was unknown until recently, when it was shown that CUG can be decoded by a specific leucyl-tRNA to initiate alternative translation in an eIF2A-dependent pathway (Starck et al., 2012). We demonstrate that such a mechanism is responsible for the synthesis of PTENα. Bioinformatics analysis predicted a Kozak-like codon context and mRNA secondary structural features for translation initiation at CUG codons (Wegrzyn et al., 2008). In this study, we found a perfect palindromic motif centered on the PTENα CUG513 start codon. Because disruption of this palindrome abolishes PTENα synthesis, the palindrome sequence surrounding CUG may therefore represent a signature motif for alternative Leu-tRNA initiation (see Figure S7F). PTENα is the first nonantigenic protein that is synthesized through the Leu-tRNA initiation mechanism. The identification of PTENα advances our understanding of protein diversity mediated by alternative translation initiation. Recently, Hopkins et al. reported a longer form of PTEN (PTEN-Long) that is secreted into adjacent cells and antagonizes PI3K/AKT signaling (Hopkins et al., 2013). This study significantly expands the functional scope of the PTEN family from intracellular to extracellular. Although PTEN-Long was predicted to have the same translation initiation codon as PTENα based on sequence inspection, there was no peptide sequence provided or verified by mass spectrometry in the report by Hopkins et al. Based on our analysis, the CUG initiation mechanism may encode several larger forms of PTEN. It is therefore important that any longer putative PTEN isoforms be verified by mass spectrometry. As multiple forms of PTEN may exist, we consider it to be most prudent to designate PTEN isoforms as a sequential series, such as α, β, or γ. Based on our observations, canonical PTEN can also promote COX activity and ATP production, although to a lesser extent as compared with PTENα. Other recent reports have also suggested that PTEN may be involved in metabolic regulation (Fang et al., 2010, Garcia-Cao et al., 2012). In particular, data from super-PTEN mice suggest that additional copies of PTEN increase mitochondrial oxidative phosphorylation and ATP production (Garcia-Cao et al., 2012). As BAC-mediated transgenesis delivers the entire Pten locus containing the coding sequence of Ptenα into the genome of the super-PTEN mice, the observed phenotype with this metabolic shift may result from additional copies of PTENα, or from a combination of PTEN and PTENα. PTENα shares the majority of its sequence with PTEN, and viewed in retrospect, current Pten knockout mouse strains are therefore essentially models of Pten and Ptenα double knockout. Thus, phenotypic deficiencies in these double knockout mice may be partially attributed to loss of Ptenα or its αN region. By utilizing TALEN-mediated somatic PTENα knockout, our study demonstrates the essential role of PTENα in mitochondrial bioenergetics and coordinated regulation of PINK1 with canonical PTEN.