A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

R. Sumanth Iyer, Lynsey Chatham, Roger Sleigh, David W. Meek (Lead / Corresponding author)

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Abstract

The PIM1 serine/threonine protein kinase mediates growth factor and survival signalling, and cooperates potently with c-MYC during tumorigenesis. PIM1 is overexpressed in many human cancers and is a promising target for drug development. PIM1 levels are regulated mainly through cytokine-induced transcription and protein degradation, but mechanisms regulating its activity and levels remain largely unexplored. Here, we show that PIM1 is modified in vitro and in cultured cells by the Small ubiquitin-like modifier (SUMO) on two independent sites: K169, within a consensus SUMOylation motif (IK169DE171) in the active site of PIM1, and also at a second promiscuous site. Alanine substitution of E171 (within the consensus motif) abolished SUMOylation, significantly increased the half-life of PIM1, and markedly reduced its ubiquitylation. Mechanistically, SUMOylation promoted ubiquitin-mediated degradation of PIM1 via recruitment of the SUMO-targeted ubiquitin ligase, RNF4. Additionally, SUMOylated PIM1 showed enhanced protein kinase activity in vitro. Interestingly, the E171A mutant was active in vitro but displayed altered substrate specificity in cultured cells, consistent with the idea that SUMOylation may govern PIM1 substrate specificity under certain contexts. Taken together, these data demonstrate that the protein kinase activity and levels of PIM1 can be regulated by covalent post-translational modification.
Original languageEnglish
Article number3598
Pages (from-to)1-14
Number of pages14
JournalScientific Reports
Volume7
DOIs
Publication statusPublished - 15 Jun 2017

Fingerprint

Sumoylation
Ubiquitin
Protein Kinases
Catalytic Domain
Substrate Specificity
Cultured Cells
Protein-Serine-Threonine Kinases
Ubiquitination
Post Translational Protein Processing
Ligases
Alanine
Proteolysis
Half-Life
Intercellular Signaling Peptides and Proteins
Carcinogenesis
Cytokines
Survival
Pharmaceutical Preparations
In Vitro Techniques
Neoplasms

Keywords

  • PIM1
  • Oncogene
  • Protein kinase
  • SUMO
  • Post-translational modification

Cite this

Iyer, R. Sumanth ; Chatham, Lynsey ; Sleigh, Roger ; Meek, David W. / A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity. In: Scientific Reports. 2017 ; Vol. 7. pp. 1-14.
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A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity. / Iyer, R. Sumanth; Chatham, Lynsey; Sleigh, Roger; Meek, David W. (Lead / Corresponding author).

In: Scientific Reports, Vol. 7, 3598, 15.06.2017, p. 1-14.

Research output: Contribution to journalArticle

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T1 - A functional SUMO-motif in the active site of PIM1 promotes its degradation via RNF4, and stimulates protein kinase activity

AU - Iyer, R. Sumanth

AU - Chatham, Lynsey

AU - Sleigh, Roger

AU - Meek, David W.

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AB - The PIM1 serine/threonine protein kinase mediates growth factor and survival signalling, and cooperates potently with c-MYC during tumorigenesis. PIM1 is overexpressed in many human cancers and is a promising target for drug development. PIM1 levels are regulated mainly through cytokine-induced transcription and protein degradation, but mechanisms regulating its activity and levels remain largely unexplored. Here, we show that PIM1 is modified in vitro and in cultured cells by the Small ubiquitin-like modifier (SUMO) on two independent sites: K169, within a consensus SUMOylation motif (IK169DE171) in the active site of PIM1, and also at a second promiscuous site. Alanine substitution of E171 (within the consensus motif) abolished SUMOylation, significantly increased the half-life of PIM1, and markedly reduced its ubiquitylation. Mechanistically, SUMOylation promoted ubiquitin-mediated degradation of PIM1 via recruitment of the SUMO-targeted ubiquitin ligase, RNF4. Additionally, SUMOylated PIM1 showed enhanced protein kinase activity in vitro. Interestingly, the E171A mutant was active in vitro but displayed altered substrate specificity in cultured cells, consistent with the idea that SUMOylation may govern PIM1 substrate specificity under certain contexts. Taken together, these data demonstrate that the protein kinase activity and levels of PIM1 can be regulated by covalent post-translational modification.

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