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Updated: Jul 11, 2026
Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line
Published on: April 13, 2022
Isolation and characterization of mouse testis specific serine/threonine kinase 5 possessing four alternatively
Youheng Wei1, Guolong Fu, Hairong Hu
1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, PR China.
Abstract:
Phosphorylation on serine/threonine or tyrosine residues of target proteins is an essential and significant regulatory mechanism in signal transduction during many cellular and life processes, including spermatogenesis, oogenesis and fertilization. In the present work, we reported the isolation and characterization of mouse testis-specific serine/threonine kinase 5 (Tssk5), which contains four alternatively spliced variants including, Tssk5alpha, Tssk5beta, Tssk5gamma and Tssk5delta. Moreover, the locus of Tssk5 is on chromosome 14qC3 and the four variants had a similar high expression in the testis and the heart; however, had a low expression in other tissues, except for Tssk5alpha which also had comparably high expression in the spleen. Each variant of Tssk5 expression began in the testis 16 days after birth. Aside from TSSK5alpha, the other isoforms have an insertion of ten amino acid residues (RLTPSLSAAG) in region VIb (HRD domain) (His-Arg-Asp). Moreover, only TSSK5alpha exhibited kinase activity and consistently, a further Luciferase Reporter Assay demonstrated that TSSK5beta, TSSK5gamma and TSSK5delta cannot be stimulated at the CREB/CRE responsive pathway in cmparison to TSSK5alpha. These findings suggest that TSSK5beta, TSSK5gamma, TSSK5delta may be pseudokinases due to the insertion, which may damage the structure responsible for active kinase activity. Pull-down assay experiments indicated that TSSK5beta, TSSK5gamma and TSSK5delta can directly interact with TSSK5alpha. In summary, these four isoforms with similar expression patterns may be involved in spermatogenesis through a coordinative way in testis.
Insights
Mouse testis-specific serine/threonine kinase 5 (Tssk5) has four variants. Only Tssk5alpha shows kinase activity, while others may be pseudokinases, potentially regulating spermatogenesis coordinately.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Protein phosphorylation is crucial for cellular signaling in reproduction.
- Testis-specific serine/threonine kinase 5 (Tssk5) plays a role in male reproductive processes.
Purpose of the Study:
- To isolate and characterize the mouse Tssk5 gene and its alternatively spliced variants.
- To investigate the expression patterns, kinase activity, and interactions of Tssk5 isoforms.
Main Methods:
- Isolation and characterization of Tssk5 variants.
- Gene expression analysis in various tissues.
- Luciferase Reporter Assay for pathway analysis.
- Pull-down assays for protein interaction studies.
Main Results:
- Identified four Tssk5 variants (Tssk5alpha, Tssk5beta, Tssk5gamma, Tssk5delta) with high expression in testis and heart.
- Only Tssk5alpha exhibited kinase activity; other variants showed impaired CREB/CRE pathway activation.
- Other isoforms possess an insertion in the HRD domain, suggesting pseudokinase function.
- Tssk5beta, Tssk5gamma, and Tssk5delta interact with Tssk5alpha.
Conclusions:
- Tssk5 isoforms exhibit distinct functional properties, with Tssk5alpha being the active kinase.
- The other isoforms may function as pseudokinases, potentially regulating Tssk5alpha activity.
- These isoforms may coordinately participate in spermatogenesis within the testis.
