||Areas of Research / rotation projects:
1) Roles for STYX in MAPK Signaling and Nuclear Transport during Male Germ Cell Maturation
The phospho-serine, -threonine or -tyrosine interaction protein (STYX) is the archetype of a unique family of "dead-phosphatases" whose members control cellular processes including cell-cycle progression, proliferation, differentiation and death. Styx / dead-phosphatases derive function through domains that structurally resemble protein tyrosine phosphatases (PTPs) except for one or more naturally occurring substitutions to amino acids that are essential for PTP catalytic activity. Rather than being the dysfunctional non-catalytic relics of active PTPs, individual STYX / dead-phosphatases act as anchors, adaptors, scaffolds or inhibitors to modulate the activity of their phosphorylated cellular targets (Wishart & Dixon, 1998). In previous work, we showed that the archetypal STYX protein was essential for male reproduction by regulating the normal maturation of germ cells in mouse (Wishart & Dixon, 2002).
Production of fertile sperm is the end product of germ cell maturation from a pool of self-renewing stem cells, followed by mitotic and meiotic division, nuclear condensation and terminal differentiation. Successful completion of each stage requires precise regulation of gene expression and nuclear protein composition. We hypothesize that STYX regulates the function of protein kinases that translocate to the nucleus to alter gene transcription and chromosomal signaling during germ cell maturation. In support of this hypothesis, we show that knockout of STYX expression in mouse results in dramatic defects of nuclear condensation in differentiating male germ cells. Chromosomal aberrations also arise in cells that are pre-meiotic and are exhibited throughout meiotic metaphase division. Moreover, STYX inhibits signaling to specific isoforms of mitogen-activated protein kinases (MAPKs) in cultured cells and directly binds phosphorylated peptides from MAPKs in vitro. Styx regulatory function is important because MAPKs are activated at several stages during germ cell maturation where they translocate to the nucleus to alter cell-cycle progression and chromatin structure. Finally, Styx physically interacts with the nuclear transport protein, Karyopherin b3 (Kpnb3), from germ cell tissue. Notably, regulation of the nucleo-cytoplasmic transport of Kpnb3 correlates with germ cell maturation and is proposed to drive cellular differentiation.
Ongoing work in the lab focuses on identifying the molecular mechanisms of STYX action in MAPK activation and modulation of nuclear signaling by import protein Kpnb3. Our long-term goal is to determine the molecular mechanism of STYX action in germ cell proliferation and differentiation as a prerequisite to defining the causes of infertility and development of intervention strategies, including the ex vivo culturing of germ cells. This research has been supported, in part, by the BIRCWH-NIH career development program (MJW).
2) Jumpy Eggs: Role for a Myotubularin-related Phosphatase in Follicular and Oocyte Development
The family of human disease-related proteins, the myotubularins (MTMs), function as D3-specific phosphatidyl-inositol (PI) phosphatases to reverse the effects of PI-3 kinases on cell signaling. We have identified the so-called egg-derived tyrosine phosphatase (EDTP/Jumpy), as a new subclass of MTM-related lipid phosphatase. EDTP/Jumpy is a maternal-effect gene during embryogenesis and is associated with sex-biased defects in neuromuscular function, resistance to oxidative stress, and reproductive aging in the fruit fly, Drosophila melanogaster. Conservation of protein sequence among metazoan EDTP/Jumpy orthologs suggests that it plays an important role in oocyte function, embryogenesis and development in higher organisms. To further determine the role of EDTP/Jumpy expression in vivo, we have obtained mice that are heterozygous for a random insertional mutation within the Edtp locus. We have verified the insertion of an exon-trapping construct within intron 9 of the Edtp gene in these mice by genomic PCR. We hypothesize that defects in EDTP function result in abnormal ovarian 3-PI signaling, dysregulation of folliculogenesis and premature ovarian failure. A comparative analysis of the follicular and oocyte growth of the three genotypes (wild-type, heterozygous, and null) has been used to determine the extent to which the loss of Edtp expression results in an abnormal reproductive phenotype. Preantral follicles were collected from 13-day old mice and cultured over a 9-day period. Follicular and ooctye growth were monitored by averaging diameter measurements taken at right angles through the center of the follicle or oocyte. Our results indicate a decrease in follicular and oocyte growth in the knockout mice. Future work focuses on the role of EDTP/Jumpy in ovarian function through detailed phenotype characterization, morphological analysis of the ovaries and hormonal studies of the adult mice. Our overarching goal is to model ovarian developmental defects seen in EDTP/Jumpy knockout mice to human reproductive disorders. This research was supported, in part, by the BIRCWH-NIH career development program (MJW).