Regulation of the oncogenic PIM1 kinase by SUMOylation
PIM1 (Proviral Integration site for Murine leukemia virus site 1) is a constitutively active serine/threonine protein kinase that is induced physiologically by the JAK-STAT and NF-κB pathways. PIM1 mediates survival signalling in cells, and contributes to the development of resistance towards chemotherapy and radiotherapy. It is also overexpressed in a wide range of haematological malignancies, and in solid tumours of the prostate, colon, breast and lung among others, which makes it an attractive drug target. However, the mechanisms regulating PIM1 kinase activity and protein levels have remained largely unexplored. Based on PIM1 protein sequence analysis, I identified the presence of a conserved motif (IK169DE171) in the active site of PIM1 that is recognised by the Small ubiquitin-like modifier (SUMO). By site-directed mutagenesis of individual lysine residues in PIM1, I showed that PIM1 is covalently modified by SUMO at he consensus K169 site, and also at a second promiscuous site. However, the K169R mutant only abolished SUMOylation at one site, but the E171A mutant abolished SUMOylation at both sites. I also showed that members of the Protein inactivator of activated STATs (PIAS) family could act as E3 SUMO ligases for PIM1. SUMOylated PIM1 showed higher protein kinase activity in vitro when Histone H3.3 was used a substrate, indicating that SUMOylation might enhance PIM1 kinase activity. Interestingly, both K169R and E171A displayed substrate specificity in vitro and when expressed in cells, suggesting that SUMOylation might govern PIM1 substrate specificity under certain contexts. Cycloheximide chase analysis revealed that both K169R and K67M (catalytically inactive) mutants of PIM1 were less stable. This can be attributed to the observation that the K169R mutant is incapable of undergoing autophosphorylation similar to the K67M mutant of PIM1. This indicates that both K169 and K67 residues are critical for the intrinsic kinase activity, and may not represent SUMO-specific effects. On the other hand, autophosphorylation of E171A was unaffected, which makes it a better mutant to study effect of SUMOylation on PIM1. The E171A mutant was more stable than wild-type PIM1 in the ycloheximide assay suggesting that SUMOylation destabilises PIM1. In support of this result, the E171A mutant also showed reduced levels of ubiquitination. Furthermore, expression of RNF4 (a polySUMO targeted E3 ubiquitin ligase) led to degradation of SUMOylated PIM1, and siRNA knockdown of RNF4 increased endogenous levels of PIM1 in cells. Taken together, the data presented here identify a novel mechanism of PIM1 degradation that is, in part, dependent on SUMOylation. SUMOylation of PIM1 also increased PIM1 activity in vitro and appears to dictate substrate specificity. Hence, further analysis of this pathway might open avenues for therapeutic intervention.
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