Human TBC1 domain-containing kinase is a class I multidomain pseudokinase.

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder caused by mutations in the TBCK gene.
• The biochemical and biophysical properties of TBCK protein are not well understood, limiting insight into TBCKE pathogenesis.

Purpose of the Study:

• To express, purify, and biochemically characterize full-length human TBCK protein.
• To investigate the catalytic activity and nucleotide-binding properties of the TBCK pseudokinase domain.

Main Methods:

• Recombinant expression of full-length human TBCK in Spodoptera frugiperda (Sf9) cells.
• Purification of TBCK protein using affinity chromatography.
• Biochemical and biophysical analyses, including nucleotide-binding assays.

Main Results:

• Successfully expressed and purified full-length human TBCK.
• The TBCK pseudokinase domain was found to be catalytically inactive.
• Absence of nucleotide binding was observed, consistent with missing key catalytic motifs (VAIK, HRD, DFG).

Conclusions:

• TBCK functions as a class I pseudokinase.
• These findings provide a foundation for future studies on TBCK's biological role and TBCKE.
• Understanding TBCK's pseudokinase nature is vital for TBCKE molecular insights.