The Glial Cell-Derived Neurotrophic Factor (GDNF)-responsive Phosphoprotein Landscape Identifies Raptor Phosphorylation Required for Spermatogonial Progenitor Cell Proliferation*

Cytokine-dependent renewal of stem cells is a fundamental requisite for tissue homeostasis and regeneration. Spermatogonial progenitor cells (SPCs) including stem cells support life-long spermatogenesis and male fertility, but pivotal phosphorylation events that regulate fate decisions in SPCs remain unresolved. Here, we described a quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of SPCs following sustained stimulation with glial cell-derived neurotrophic factor (GDNF), an extrinsic factor supporting SPC proliferation. Stimulated SPCs contained 3382 identified phosphorylated proteins and 12141 phosphorylation sites. Of them, 325 differentially phosphorylated proteins and 570 phosphorylation sites triggered by GDNF were highly enriched for ERK1/2, GSK3, CDK1, and CDK5 phosphorylating motifs. We validated that inhibition of GDNF/ERK1/2-signaling impaired SPC proliferation and increased G2/M cell cycle arrest. Significantly, we found that proliferation of SPCs requires phosphorylation of the mTORC1 component Raptor at Ser863. Tissue-specific deletion of Raptorin mouse germline cells results in impaired spermatogenesis and progressive loss of spermatogonia, but in vitroincreased phosphorylation of Raptor by raptor over-expression in SPCs induced a more rapidly growth of SPCs in culture. These findings implicate previously undescribed signaling networks in governing fate decision of SPCs, which is essential for the understanding of spermatogenesis and of potential consequences of pathogenic insult for male infertility.