Protein kinases, the PIM story

Last week I was presenting my DPhil work. In one of my projects I address the reasons for inhibitor selectivity in PIM protein kinase family. PIM kinases play key roles in signalling pathways and have been identified as oncogenes long time ago. Slightly unusual for protein kinases ATP-binding sites and cancer roles have prompted the investigation of potential PIM-selective inhibitors for anticancer therapy. Due to overlapping functions of the three PIM isoforms, efficacious inhibitors should bind to all three isozymes. However, most reported inhibitors show considerable selectivity for PIM1 and PIM3 over PIM2 and the mechanisms leading to this selectivity remain unclear.

Figure 1. Workflow of the sequence and structure analysis of PIM kinases

Figure 1. Workflow of the sequence and structure analysis of PIM kinases

To establish the sequence determinants of inhibitor selectivity we investigated the phylogenetic relationships of PIM kinases and their structural conformations upon ligand binding (Figure 1). Together with my OPIG supervisor Charlotte Deane we predicted a set of candidates for site-directed mutagenesis as illustrated in Figure 2. The mutants were designed to convert PIM1 residues into analogous PIM2 residues at the same positions.

I then moved to the wetlab to test the hypotheses experimentally. Under guidance of Oleg Fedorov, I screened the SGC library of kinase inhibitors using differential scanning fluorimetry (DSF). After comparing melting temperature shift values across the PIM kinases and mutants, a set of potent inhibitors with different chemical scaffolds have been selected for quantitative binding analysis. I worked with Peter Drueker’s team at Novartis on PIMs enzymology, where I measured activities, Km values for ATP and IC50s using mobility shift assay. For my final set of measurements I performed isothermal titration calorimetry (ITC) experiments back at the SGC and determined binding constants and enthalpic/entropic contributions to the total free energy of ligand binding.

Figure 2. An overlay of PIM1 and PIM2 structures (P-loop and hinge regions), the mutated residues are shown as sticks

Figure 2. An overlay of PIM1 and PIM2 structures (P-loop and hinge regions), the mutated residues are shown as sticks

The data are yet to be published, I only briefly state the results here. The hinge mutant E124L demonstrated reduced thermal stability probably due to removal of E124-R122 salt bridge. The P-loop mutants had intermediate Km ATP values between PIM1 and PIM2, indicating that those residues could be responsible for stronger ATP binding in PIM2. As shown in Figure 2, the residues are located at the tip of the P-loop and might have involvement in the P-loop movement. Importantly, three mutants have shown reduced affinity to inhibitors validating my initial hypotheses.

Ideally having PIM1 and PIM2 co-crystal structures with the same inhibitors would allow direct comparison of the binding modes. So far I was able to solve apo-PIM2 structure in addition to the single PIM2 pdb, which will be deposited shortly.

I will update you soon about on my second project which involves more mutants, type II inhibitors, equilibrium shifts and speculations about conformational transitions. Keep visiting us!

3 thoughts on “Protein kinases, the PIM story

  1. Pingback: [Publication] Effect of Single Amino Acid Substitution Observed in Cancer on Pim-1 Kinase Thermodynamic Stability and Structure | Oxford Protein Informatics Group

  2. justin moore

    As far as I understand it, there are three isoforms of Pim2. Also, I am currently looking over a paper published in April 2012 (Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2) in which they state an opposite effect between the 34 KDa and 41 KDa isoforms (the smaller seeming to prevent cell proliferation while the larger has potent anti-apoptotic and cell proliferation effects). I have a couple questions with this in mind. Do you know, specifically, which isoform crystal structure people are working with? The stuff I come across simply mentions Pim-2 structure as a single entity but I am wondering if there is a predominance of one isoform being crystalized over other isoforms of Pim-2. Considering that it has been published that different isoforms have different functions, would it not be advantageous to be specific as to which isoform is being crystalized or are the isoforms too closely related to be differentiated? Thanks for your time.

    Regards,
    Justin Moore

    1. Leila Alexander Post author

      Thank you for your interest, Justin.
      PIM2 protein is not one to crystallise easily and so far there is only one publically available structure (PDB:2IWI), which was obtained by expressing the full length 34 kDa PIM2 (Uniprot:Q9P1W9). The N-terminus residues in this structure were not defined by the electron density and are very likely to be disordered. In my project, I crystallised PIM2 using a truncated version of this construct, and the new structures are not yet deposited to the PDB.

      Like in the case with PIM1, the longer isoform could be potentially recruited to the plasma membrane, thereby defining the substrate specificity (Brault et. al. 2010). However, since the kinase domains are identical in long and short isoforms (as they result due to the use of alternative initiation sites), it is fair to assume that there is no difference in the inhibition of the ATP-binding site.
      I hope this helps.
      Leila

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