Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase

Abstract

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca 2؉ channels are already partially activated in vehicletreated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicletreated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca 2؉ channel blocker, and chelation of extracellular Ca 2؉ . These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.The cytoplasmic p42/p44 mitogen-activated protein kinase (MAPK) 1 /extracellular signal-regulated kinases (ERKs) comprise a critical convergence point in the signaling pathways initiated by a variety of receptor agonists that promote cellular differentiation or proliferation. For the classic receptor tyrosine kinase-initiated pathway, growth factors like epidermal growth factor (EGF) induce the autophosphorylation of their receptors and create specific binding sites for Src homology 2-containing proteins such as Grb2 (1). Grb2 complexed to Sos associates with the receptor tyrosine kinase, and Sos stimulates GDP release from Ras, leading to Ras activation. Active Ras then binds to Raf-1, leading to its activation, and Raf-1 in turn catalyzes the serine phosphorylation and activation of the MAPK/ERK kinase MEK. MEK then catalyzes the phosphorylation of ERK on regulatory Thr and Tyr residues, resulting in ERK activation.Guanine nucleotide-binding protein-coupled receptors (GPCRs) are also well known activators of ERK; however, there are a variety of pathways by which GPCRs promote ERK activation. Often, GPCRs such as those activated by lysophosphatidic acid or angiotensin II promote the transactivation of a receptor tyrosine kinase as evidenced by its increased tyrosine phosphorylation (2). Receptor tyrosine kinase transactivation directs the tyrosine phosphorylation of adaptor proteins such as Shc, recruitment of the Grb2-Sos complex, and subsequent Ras activation. It is less clear how GPCRs promote the tyrosine phosphorylation of the receptor tyrosine kinase, although Src activation downstream of the G␤␥ has been implicated in some cells (3, 4). For those GPCRs whose activated G␣ subunits promote...

Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation

“…These results suggest that PP1 is necessary for FSH to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R. The ability of FSH to promote the dephosphorylation of MLC(Ser 19 ) additionally indicates that FSH activates PP1.…”

“…Results (Fig. 5B) show that pretreatment of GCs with H89 did not inhibit -fold FSH-stimulated activation of PP1, as evidenced by dephosphorylation of MLC(Ser 19 ). This result supports the contribution of PP1 to FSH-dependent IRS1(Tyr 989 ) phosphorylation.…”

“…8D) ) was a positive control. Furthermore, transduction of GCs with the constitutively active PKA catalytic subunit mutant PKA-CQR was sufficient to stimulate MLC(Ser 19 ) dephosphorylation (Fig. 5A).…”

“…Tautomycin, but not okadaic acid, blocked FSH-stimulated IRS1(Tyr 989 ) and AKT(Thr 308 ; Ser 473 ) phosphorylations. Myosin light chain (MLC)(Ser 19 ) is a direct target of PP1 (59), and glycogen synthase kinase 3␤(Ser 9 ) is a direct target of PP2 (62); both serve as positive controls for inhibitor efficacy. These results suggest that PP1 is necessary for FSH to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R.…”

“…FSH binds its GPCR, leading to the activation of PKA via adenylyl cyclase-mediated cAMP production (18). Transcriptional responses to FSH are dependent upon activation of PKA, based on the ability of the catalytic inhibitor PKI or H89 to inhibit target gene expression in cultured GCs (9,13,19,20). Likewise, lentiviral expression of a constitutively active PKA catalytic subunit mutant (PKA-CQR) closely mimics gene expression patterns of FSH (21), suggesting that PKA is necessary and sufficient for most transcriptional actions of FSH.…”