PARENT SESSION

(M745) FSH ELICITS C-TERMINAL PHOSPHORYLATION OF THE SERINE THREONINE KINASE LKB1.

Feiger, Zachary¹, Maizels, Evelyn¹, Flynn, Maxfield¹, Hunzicker-Dunn, Mary ¹, ¹ Feinberg School of Medicine, Northwestern University, Chicago, IL

ABSTRACT- FSH regulates the differentiation of granulosa cells by activation of a network of signaling events. FSH binds to its cognate receptor to activate adenylate cyclase, resulting in elevation of cyclic AMP levels and activation of protein kinase A (PKA) in granulosa cells. A recently identified target of PKA is the serine threonine kinase LKB1, also known as STK11, which has been identified to be the key gene product mutated in the Peutz-Jeghers syndrome, a cancer susceptibility syndrome with disordered growth . LKB1 has been identified to act in concert with the pseudokinase STRAD and binding partner MO25 to control rates of cell growth as well as polarity of cells through the phosphorylation of an extended family of target kinases comprised of kinases closely or more distantly related to AMP-activated protein kinase. PKA is reported to phosphorylate a key serine residue close to the C-terminus of LKB1 which lies within a cognate PKA target motif, RRXS/T, corresponding to serine 428 in the human LKB1 sequence. Serine 428 phosphorylation has been characterized to be required for function of LKB1, as mutation of this site to non-phosphorylatable residues will block the growth inhibitory phenotype attributed to LKB1 activation. We investigated whether FSH treatment of immature rat granulosa cells might elicit the phosphorylation of the C-terminal serine residue in rat LKB1. Immature granulosa cells in primary culture were treated with FSH, and total cell extracts were prepared and subjected to immunoblot analysis using phospho-specific antibody recognizing the phosphorylated serine 428 of LKB1. Two bands were detected with the phospho-specific antibody, one corresponding to a molecular weight of 50 kD consistent with the molecular weight of LKB1 reported in other species, and an additional band corresponding to a molecular weight of 60 kD, which is consistent with the related rat serine threonine kinase designated as similar to serine/threonine-protein kinase LKB1 (accession XM 234900.1). Both bands were increased by one hour in response to FSH treatment, indicating that FSH increased phosphorylation of the c-terminal serines in both forms of LKB1. These results suggest that LKB1 is a novel FSH target in granulosa cells with the potential to regulate differentiation or cell polarty. Supported by HD21921

KEY WORDS: FSH, granulosa, serine threonine kinase, LKB1