PARENT SESSION

(T773) PTP EPSILON CORRESPONDS TO THE 100 kDa PROTEIN TYROSINE PHOSPHATASE THAT ASSOCIATES WITH ERK AND REGULATES FSH DEPENDENT ERK ACTIVITY IN PREANTRAL GRANULOSA CELLS.

Peters, Carl¹, Maizels, Evelyn¹, Elson, Ari², Hunzicker-Dunn, Mary¹, ¹ Northwestern University, Chicago, IL² The Weizmann Institute of Science, Rehovot, Israel

ABSTRACT- We have previously reported in vehicle-treated rat preantral granulosa cells that the protein kinase upsteam of p42/p44 mitogen activated protein kinase/extracellular regulated kinase (ERK) is already activated but that a protein tyrosine phosphatase (PTP) associated with ERK maintains ERK in an inactive state (Cottom et al., 2003). We have also reported that treatment of granulosa cells with follicle stimulating hormone (FSH) leads to phosphorylation of this PTP by protein kinase A (PKA) and consequent dissociation of ERK, resulting in ERK activation . While this granulosa cell PTP reacted with one antiserum directed towards PTP-SL, the granulosa cell PTP did not correspond to PTP-SL, based on size and lack of reactivity to a panel of PTP-SL antibodies. Our current work has investigated whether this phosphatase corresponds to PTP epsilon, a phosphatase which has previously been shown to associate with and inhibit ERK activity in NIH3T3 cells. PTP epsilon has a potential PKA phosphorylation site at threonine 433 in the rat sequence that is conserved in mice and humans (T433 and T434 respectively). Western blotting with a PTP epsilon antibody showed that it is indeed expressed in granulosa cells and migrates at 97 kD, the size of the granulosa cell PTP associated with ERK. In addition following an ERK agarose pull-down, western blotting with the PTP epsilon antibody showed that PTP epsilon is indeed associated with ERK and that FSH stimulation decreases the amount of PTP epsilon that is associated, congruent with our previous results. Furthermore western blotting results with the PTP epsilon antibody following immune-precipitation with an antibody that detects the phosphorylated PKA substrate motif indicate that PTP epsilon is indeed a PKA substrate and that phosphorylation of PTP epsilon is stimulated by FSH. In summary our current results indicate that PTP epsilon is involved in regulation of ERK activity downstream of FSH. In unstimulated granulosa cells PTP epsilon is associated with ERK giving it the opportunity to repress ERK activity. Following FSH stimulation phosphorylation of PTP epsilon by PKA relieves ERK of its association with PTP epsilon thus freeing ERK to become active. Supported by HD 21921.

KEY WORDS: protein tyrosine phosphatse epsilon, mitogen activated protein kinase, granulosa cells, follicle stimulating hormone