Endocrine Abstracts

Introduction: Prolactin (PRL) is a hormone utilized at both endocrine and autocrine levels. PRL regulates cyclin D1 expression through JAK2-mediated transcriptional activation of cyclin D1 promoter. Serine–threonine kinase PAK1 is also implicated in regulation of cyclin D1 gene expression. We previously showed that PAK1 binds to and is Tyr phosphorylated by JAK2. Herein we linked PRL and PAK1 as JAK2 substrate to stimulation of cyclin D1 promoter activity.

Methods: Two-dimensional peptide mapping and LC–MS/MS mass spectrometry identified three Tyr(s) of PAK1 (153, 201 and 285) which are phosphorylated by JAK2. We measured induction of cyclin D1 promoter activity in T47D cells expressing cyclin D1 promoter-luciferase and either PAK1 WT or PAK1 Y3F (three tyrosines are mutated). We used confocal immunofluorescence and cell fractionation to study a role of PAK1 nuclear localization for cyclin D1 promoter regulation.

Results: PAK1 activates cyclin D1 promoter in response to PRL. Mutation of JAK2 phosphorylation sites on PAK1 decreases both PRL-induced PAK1 nuclear translocation and cyclin D1 promoter activity by 55%. Mutation of PAK1 nuclear-localization signals (NLS) decreases PRL-induced cyclin D1 promoter activity by 46%. A PAK1 Y3F mutant lacking NLSs decreases cyclin D1 activity by 68% suggesting that there is another PAK1-dependent mechanism to activate cyclin D1 promoter. We found that adapter protein Nck sequesters PAK1 in cytoplasm and co-expression of PAK1 and Nck inhibits amplifying effect of PRL-induced PAK1 on cyclin D1 promoter activity (95% inhibition). This inhibition is partially abolished by disruption of PAK1-Nck binding.

Conclusion: We propose two PAK1-dependent mechanisms to activate cyclin D1 promoter activity in response to PRL: via nuclear translocation of Tyr-phosphorylated PAK1 and via formation of Nck-PAK1 complex that sequesters PAK1 in cytoplasm.

Declaration of interest: I fully declare a conflict of interest. Details below.

Funding: This work was supported however funding details unavailable.

R01 NIH/NIDDK, DK 088127.