Evolutionary and cellular analysis of the 'dark' pseudokinase PSKH2

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Pseudokinases, including human PSKH2, are related to active kinases but lack catalytic function.
• Vertebrate PSKH2 proteins' biological roles are largely unknown, despite evolutionary adaptations.
• PSKH2 is a pseudokinase paralog to the canonical kinase PSKH1.

Purpose of the Study:

• To characterize vertebrate PSKH2 orthologues using structural informatics, biochemistry, and proteomics.
• To elucidate the functional roles of PSKH2's N- and C-terminal regions.
• To understand the regulation and cellular localization of human PSKH2.

Main Methods:

• AlphaFold 2-based structural analysis.
• Biochemical assays to confirm lack of phosphotransferase activity.
• Cellular truncation analysis.
• Mass spectrometry-based proteomics.
• Analysis of HSP90/Cdc37 chaperone interactions.

Main Results:

• PSKH2 lacks detectable protein phosphotransferase activity.
• The N-terminal domain is essential for PSKH2 expression and mitochondrial localization.
• Human PSKH2 is part of a mitochondrial protein network.
• PSKH2 expression is regulated by the HSP90/Cdc37 chaperone system.
• The C-terminal tail mediates HSP90 interactions, suggesting a regulatory role.

Conclusions:

• PSKH2 functions as a pseudokinase, with its N-terminal domain critical for stability and mitochondrial targeting.
• The C-terminal tail's interaction with HSP90 suggests a regulatory role in signaling pathways.
• PSKH2 is integrated into mitochondrial protein networks and regulated by molecular chaperones.