Systematic Analysis of the Phosphoproteome and Kinase-substrate Networks in the Mouse Testis

Abstract

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 TiO 2 , 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 kinasesubstrate 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 sitespecific 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. Molecular & Cellular Proteomics 13:

Valproic acid changes the expression of tyrosine‐phosphorylated proteins in rat seminal vesicle

“…Many studies have also reported that VPA can cause decreases in sex hormones, including testosterone (Hamza & Amin, ; Ocek et al, ; Sukhorum & Iamsaard, ; Wasilewska‐Dziubinska et al, ; Zhao et al, ), which is crucial to the development process and regulation of micronutrient secreting activity of the seminal vesicle, especially in mammals (Higgins & Burchell, ; Tao, Lei, & Rao, ). This hormonal decrease has been explained by the fact that VPA treatment leads to low expressions of androgen receptor (AR) and cholesterol side‐chain cleavage enzyme (P450scc) in rats (Qi et al, ; Sukhorum & Iamsaard, ). Consequently, adverse effects may result, such as seminal atrophy, histopathological alterations and secretion reduction (including biochemical parameters), as shown in this study.…”

“…Such proteins are modified by phosphorylation on tyrosine residue of translated proteins. TyrPho proteins are involved in cellular proliferation, division, growth and differentiation (Koromilas, ; Qi et al, ; Wang, Peterson, & Loring, ). Originally, TyrPho proteins were demonstrated to have essential roles in sperm capacitation and the acrosome reaction process, both in vivo and in vitro (Buffone, Kim, Doak, Rodriguez‐Miranda, & Gerton, ; Kwon, Rahman, & Pang, ; Naz & Rajesh, ; Romarowski et al, ).…”

In Silico Tools and Phosphoproteomic Software Exclusives

“…In vitro methods are not effective in reflecting in vivo activities of kinases, which is why MS-based methods are needed for evaluating kinase activity [82,83]. An approach to link phosphoproteomics data with the activity of kinases was presented by Qi et al [84], which is known as kinase activity analysis (KAA). CLUE (CLUster Evaluation) is a method designed specifically for phosphoproteomics data [85], based on the hypothesis that phosphosites targeted by the same kinase will show similar temporal profiles.…”

Phosphoproteomics-based Profiling of Kinase Activities in Cancer Cells

“…Another approach to link phosphoproteomics data with the activity of kinases was presented in a publication from Qi et al [Qi et al, 2014], which they termed kinase activity analysis (KAA). In this study, the authors collected phosphoproteomics data from adult mouse testis in order to investigate the process of mammalian spermatogenesis.…”

“…As a means to extract functional insight, methods to infer kinase activities from phosphoproteomics data based on prior-knowledge about kinase-substrate relationships have been put forward [Qi et al, 2014;Casado et al, 2013;Yang et al, 2015;Mischnik et al, 2015]. The knowledge about kinase-substrate relationships, compiled in databases like PhosphoSitePlus [Hornbeck et al, 2015] or Phospho.ELM [Dinkel et al, 2011], covers only a limited set of interactions.…”