Date Published: June 5, 2013
Publisher: Public Library of Science
Author(s): Clorinda Lori, Antonella Lantella, Alessandra Pasquo, Leila T. Alexander, Stefan Knapp, Roberta Chiaraluce, Valerio Consalvi, Annalisa Pastore. http://doi.org/10.1371/journal.pone.0064824
Pim-1 kinase, a serine/threonine protein kinase encoded by the pim proto-oncogene, is involved in several signalling pathways such as the regulation of cell cycle progression and apoptosis. Many cancer types show high expression levels of Pim kinases and particularly Pim-1 has been linked to the initiation and progression of the malignant phenotype. In several cancer tissues somatic Pim-1 mutants have been identified. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of Pim-1 kinase. We expressed and purified some of the mutants of Pim-1 kinase that are expressed in cancer tissues and reported in the single nucleotide polymorphisms database. The point mutations in the variants significantly affect the conformation of the native state of Pim-1. All the mutants, expressed as soluble recombinant proteins, show a decreased thermal and thermodynamic stability and a lower activation energy values for kinase activity. The decreased stability accompanied by an increased flexibility suggests that Pim-1 variants may be involved in a wider network of protein interactions. All mutants bound ATP and ATP mimetic inhibitors with comparable IC50 values suggesting that the studied Pim-1 kinase mutants can be efficiently targeted with inhibitors developed for the wild type protein.
Pim-1 kinase belongs to a family of serine/threonine protein kinases (EC 188.8.131.52) that are encoded by the pim proto-oncogenes –. Pim-1 locus has been originally identified as a common Proviral insertion site in moloney murine leukemia virus-induced T-cell lymphomas in mice . The encoded protein kinase is involved in several signalling pathways such as the regulation of cell cycle progression and apoptosis. The three Pim family members Pim-1, Pim-2 and Pim-3 identified in humans have been reported as signalling protein kinases playing an important role in tumor biology , . In addition, many cancer types show high expression levels of Pim kinases. For instance Pim-1 and Pim-2 have been reported to be highly expressed in leukemia, lymphoma, prostate cancer and multiple myeloma and are considered to be involved in the initiation and progression of the malignant phenotype . In addition, Pim-1 has been identified as a key cofactor regulating the expression of the oncogenic transcription factor c-Myc by phosphorylating serine 10 in histone H3 in enhancer region of the Myc locus and its target genes .
This study represents, to our knowledge, the first spectroscopic and thermodynamic characterization of human kinase Pim-1 and some of its disease relevant mutants found in cancer. We investigated the effect of amino acid substitution on the thermal and thermodynamic stability of wild type Pim-1 and compared these results with four Pim-1 variants, Y53H, E124Q, E135K and E142D, reported in SNPs database –, , . Examination of the Pim-1 crystal structure showed that most mutations are located close to structural elements important for kinase function and that form polar interactions with neighbouring residues.