Systematic analysis of the phosphoproteome and kinase-substrate networks in the mouse testis

Mol Cell Proteomics. 2014 Dec;13(12):3626-38. doi: 10.1074/mcp.M114.039073. Epub 2014 Oct 7.


Spermatogenesis is a complex process closely associated with the phosphorylation-orchestrated cell cycle. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3955 phosphoproteins. Although only approximately half of the phosphorylation sites enriched by IMAC were also captured by TiO2, both the phosphoprotein data sets identified by the two methods significantly enriched the functional annotation of spermatogenesis. Thus, the phosphoproteome profiled in this study is a highly useful snapshot of the phosphorylation events in spermatogenesis. To further understand phosphoregulation in the testis, the site-specific kinase-substrate relations were computationally predicted for reconstructing kinase-substrate phosphorylation networks. A core sub-kinase-substrate phosphorylation networks among the spermatogenesis-related proteins was retrieved and analyzed to explore the phosphoregulation during spermatogenesis. Moreover, network-based analyses demonstrated that a number of protein kinases such as MAPKs, CDK2, and CDC2 with statistically more site-specific kinase-substrate relations might have significantly higher activities and play an essential role in spermatogenesis, and the predictions were consistent with previous studies on the regulatory roles of these kinases. In particular, the analyses proposed that the activities of POLO-like kinases (PLKs) might be dramatically higher, while the prediction was experimentally validated by detecting and comparing the phosphorylation levels of pT210, an indicator of PLK1 activation, in testis and other tissues. Further experiments showed that the inhibition of POLO-like kinases decreases cell proliferation by inducing G2/M cell cycle arrest. Taken together, this systematic study provides a global landscape of phosphoregulation in the testis, and should prove to be of value in future studies of spermatogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CDC2 Protein Kinase / genetics
  • CDC2 Protein Kinase / metabolism
  • Cell Cycle Proteins / antagonists & inhibitors
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cyclin-Dependent Kinase 2 / genetics
  • Cyclin-Dependent Kinase 2 / metabolism
  • G2 Phase Cell Cycle Checkpoints / drug effects
  • G2 Phase Cell Cycle Checkpoints / genetics
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Gene Regulatory Networks*
  • Male
  • Mice
  • Mitogen-Activated Protein Kinases / genetics
  • Mitogen-Activated Protein Kinases / metabolism
  • Molecular Sequence Annotation
  • Phosphoproteins / genetics*
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Polo-Like Kinase 1
  • Protein Kinase Inhibitors / pharmacology
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Proteome / genetics*
  • Proteome / metabolism
  • Proto-Oncogene Proteins / antagonists & inhibitors
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Spermatogenesis / genetics*
  • Testis / cytology
  • Testis / enzymology*


  • Cell Cycle Proteins
  • Phosphoproteins
  • Protein Kinase Inhibitors
  • Proteome
  • Proto-Oncogene Proteins
  • Protein Serine-Threonine Kinases
  • CDC2 Protein Kinase
  • Cdk2 protein, mouse
  • Cyclin-Dependent Kinase 2
  • Mitogen-Activated Protein Kinases