Originally discovered as a metastasis suppressor, KISS1 has since been defined as a neurotransmittor and regulator of diverse cellular functions and has been implicated in pathologies such as hypogonadism and Alzheimer's disease. Several laboratories are actively developing therapies based upon KISS1 biology. KISS1 is secreted and processed into small peptides (called kisspeptins (KP)) in the extracellular milieu. However, the enzyme(s) responsible for generation of KP from nascent KISS1 is unknown, although sequence analysis (cleavage at KR or RR dibasic sites) suggests that proprotein convertases (PC) are responsible. We hypothesized that enzyme(s) belonging to the PC family processes KISS1 to generate KP. KISS1 processing was completely inhibited by treatment with PC inhibitors Dec-RVKR-CMK and a1-PDX in these cells multiple tumor cell lines. Further, mRNA expression of the seven members of the PC family showed consistent expression of only three proteases - furin, PCSK5 and PCSK7 narrowing the candidate proteases. Only shRNA-mediated knockdown of furin (i.e., not PCSK5 or PCSK7) blocked KISS1 processing. Thus furin is the essential enzyme responsible for KP generation. Even when cells were exposed to Dec-RVKR-CMK or shRNA targeting furin, two bands (Mr∼15.9 kDa and Mr ∼17 kDa) were consistently detected in SDS-PAGE of conditioned medium. This observation suggested KISS1 could either be alternatively post-translationally modified or that another protease (family) might also cleave KISS1. To investigate the possibility that KISS1 may be post-translationally modified, potential sites of O-glycosylation (NetOGlyc 4.0) or serine/threonine phosphorylation (NetPhos 2.0) were analyzed in silico. Three amino acid residues (T69, S70, and S72)of the native KISS1 protein showed highest scores as being either O-glycosylated or phosphorylated. Further, mutation of these three residues to alanine resulted in the loss of the higher molecular weight band which was co-localized to a band detected using anti-phospho Ser/Thr strongly suggesting that KISS1 is indeed phosphorylated. This is the first report of phosphorylation of KISS1. Studies are underway to identify the kinase(s) and phosphatase(s) involved as well as the functional significance of the phosphorylation. Support: CA134981, National Foundation for Cancer Research, Steiner Family Fellowship in Metastasis Research, Susan G. Komen for the Cure SAC110037.
Citation Format: Sitaram Harihar, Kelsey R. Hampton, Tomoo Iwakuma, Nabil G. Seidah, Danny R. Welch. Phosphorylation and furin-mediated processing are critical posttranslational modifications of the KISS1 metastasis suppressor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 135. doi:10.1158/1538-7445.AM2014-135