Reproductive function in protein kinase inhibitor-deficient mice.

The protein kinase inhibitor (PKI) family includes three genes encoding small, heat-stable inhibitors of the cyclic AMP-dependent kinase PKA. Each PKI isoform contains a PKA inhibitory domain and a nuclear export domain, enabling PKI to both inhibit PKA and remove it from the nucleus. The PKIbeta isoform, also known as testis PKI, is highly expressed in germ cells of the testis and is found at more modest levels in other tissues. In order to investigate its physiological role, we have generated PKIbeta knockout mice by gene targeting. These mice exhibit a partial loss of PKI activity in testis but remain fertile with normal testis development and function. PKIbeta knockout females also reproduce normally. The PKIbeta mutants were crossed with our previously derived PKIalpha mutants to obtain double-knockout mice. Remarkably, these mice are also viable and fertile with no obvious physiological defects in either males or females.

Deficient gene expression in protein kinase inhibitor alpha Null mutant mice.

Protein kinase inhibitor (PKI) is a potent endogenous inhibitor of the cyclic AMP (cAMP)-dependent protein kinase (PKA). It functions by binding the free catalytic (C) subunit with a high affinity and is also known to export nuclear C subunit to the cytoplasm. The significance of these actions with respect to PKI's physiological role is not well understood. To address this, we have generated by homologous recombination mutant mice that are deficient in PKIalpha, one of the three isoforms of PKI. The mice completely lack PKI activity in skeletal muscle and, surprisingly, show decreased basal and isoproterenol-induced gene expression in muscle. Further examination revealed reduced levels of the phosphorylated (active) form of the transcription factor CREB (cAMP response element binding protein) in the knockouts. This phenomenon stems, at least in part, from lower basal PKA activity levels in the mutants, arising from a compensatory increase in the level of the RIalpha subunit of PKA. The deficit in gene induction, however, is not easily explained by current models of PKI function and suggests that PKI may play an as yet undescribed role in PKA signaling.

Neurotransmitters activate the human estrogen receptor in a neuroblastoma cell line.

The human neuroblastoma cell line SK-N-SH has been used as a model system to study the interactions of the human estrogen receptor (hER) with neurotransmitters. We have successfully transfected these cells using an adenoviral delivery system and have reconstituted ligand-dependent responses to estradiol and ligand-independent responses to a series of dopamine D1 receptor agonists. The full agonist for the D1 receptor, SKF 82958, shows a robust activation of hER, comparable to that induced by estradiol. This activation is blocked by the protein kinase A inhibitor H-89, is mimicked by forskolin, and is therefore thought to be mediated in part through the cAMP/protein kinase A pathway. We have examined deletion mutants of hER for activation by SKF 82958 and find that both its transactivation domains, AF-1 and AF-2, must cooperate to impart the full response to the agonist. Significantly, an agonist of the muscarinic acetylcholine receptor, carbachol, though not active by itself, synergistically activates hER in conjunction with suboptimal doses of SKF 82958. This is the first reported instance of two neurotransmitters synergizing to activate a member of the nuclear receptor superfamily, and might predict a role for multiple neural inputs modulating the effects of these receptors in the central nervous system.