Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

Voltage sensitive phosphoinositide phosphatases of Xenopus: their tissue distribution and voltage dependence.

Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.

Microinjection of follicle-enclosed mouse oocytes.

The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.

Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte.

Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from approximately 2 microM to approximately 80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from approximately 1 microM to approximately 40 nM) relieves the inhibition of PDE3A, increasing its activity by approximately 5-fold. This causes a decrease in oocyte cAMP (from approximately 700 nM to approximately 140 nM), leading to the resumption of meiosis.

CD13/APN transcription is induced by RAS/MAPK-mediated phosphorylation of Ets-2 in activated endothelial cells.

CD13/aminopeptidase N (CD13/APN) is a potent regulator of angiogenesis both in vitro and in vivo and transcription of CD13/APN in endothelial cells is induced by angiogenic growth factors via the RAS/MAPK pathway. We have explored the nuclear effectors downstream of this pathway that are responsible for CD13/APN induction. The response to serum/angiogenic growth factors mapped to a 38-bp region of the CD13/APN promoter containing an Ets-core motif that specifically binds a protein complex from nuclear lysates from activated endothelial cells. This motif and the proteins that target it are functionally relevant because mutation of this sequence abrogates CD13/APN transcription. Analysis of endothelial Ets family members showed that Ets-2, and to a lesser extent Ets-1, transactivate CD13/APN promoter activity via the Ets-core motif, whereas Fli, Erg, and NERF are ineffective. We investigated the possibility that the induction of CD13/APN is mediated by phosphorylation of Ets-2 via RAS/MAPK. A phosphorylation-defective Ets-2 mutant, T72A, failed to transactivate CD13/APN, suggesting that Ets-2 phosphorylation is obligatory for CD13/APN induction. To confirm a role for endogenous Ets-2 in CD13/APN expression, we specifically abrogated Ets-2 mRNA and protein by siRNA knockdown that significantly inhibited CD13/APN transcription. Finally, to assess the relevance of Ets-2 in endothelial cell function, we induced endothelial cells containing Ets-2 siRNA oligonucleotides to form capillary networks. Cells containing the Ets-2 inhibitory small interfering RNAs were completely incapable of forming the organized networks characteristic of endothelial morphogenesis. Thus, the phosphorylation of Ets-2 by RAS/MAPK is a prerequisite for CD13/APN endothelial induction and Ets-2 and its targets play essential roles in endothelial cell function.