Our lab is interested in understanding the mechanisms controlling eukaryotic cell growth and proliferation.  Our primary focus has been on the G0-like resting states that cells enter when conditions are not conducive to continued growth.  The transitions between active division and these periods of quiescence are known to be key points of proliferative control.  We are interested in characterizing the signaling pathways that control these transitions, and the maintenance of cell viability in the absence of cell division.  The long-term goals of this work are to develop a more complete understanding of the general control of cell proliferation, and to identify novel targets for the treatment of proliferative disorders, such as human cancer.

For these studies, we have been using the budding yeast, Saccharomyces cerevisiae.  This model organism is ideal for this work because of the powerful genetic and genomic strategies that can be readily applied to this problem.  Moreover, previous work has demonstrated that the mechanisms governing growth are highly conserved, and many of the pathways were initially worked out through studies with this yeast.  We are primarily interested in characterizing the signaling pathways that allow growth to be properly coordinated with signals in the extracellular environment.  To date, our work has focused on the cAMP-dependent protein kinase, PKA and its role in the regulation of cell growth.  We are interested in identifying the targets of this enzyme that are critical for this control, and in understanding how PKA activity is coordinated with other signaling pathways involved in the regulation of cell growth.  The following sections briefly describe the three primary areas of research being pursued in the lab.

SUBSTRATE RECOGNITION BY PROTEIN KINASES

Understanding the biological role of any protein kinase requires the identification of the substrates of that enzyme.  For these purposes, we have developed a number of novel strategies for substrate identification, and have used these to identify more than 50 targets for the S. cerevisiae PKA.  One of these approaches involves the use of PKA variants that exhibit a stable binding to substrates.  Most wild-type protein kinases, including PKA, bind only transiently to their targets.  Using these variants, we identified domains in both PKA and substrates that are important for phosphorylation in vivo.  Interestingly, these studies have suggested that substrate phosphorylation and release might be coupled, and that this coupling may be a general property of this enzyme family.  We are presently using a combination of genetic and structural methods to examine these substrate-binding forms of PKA with the goal of obtaining insights into the catalytic mechanism of these important signaling molecules.

PKA REGULATION OF AUTOPHAGY 

The above work suggested that PKA signaling is involved in the regulation of a diverse set of physiological processes.  We have been examining several of these including the highly-conserved process of macroautophagy (hereafter referred to as autophagy).  Autophagy is a membrane trafficking pathway that is responsible for the degradation of cytoplasmic protein and organelles.  This pathway is essential for cell survival during G0 arrest, and has been implicated in a variety of human disorders, including cancer and Huntington’s disease.  Our work has shown that PKA is an important regulator of autophagy in S. cerevisiae, and that the Atg1 complex is the primary target of this control.  Atg1 is a conserved protein kinase that appears to be a key regulator of autophagy in all eukaryotic cells.  Our present work is aimed at understanding how PKA phosphorylation influences Atg1 activity and at identifying the physiologically-relevant substrates of Atg1 kinase activity. 

COORDINATION OF THE PKA AND TOR SIGNALING PATHWAYS 

Cell growth is controlled by multiple signaling pathways whose activities are coordinated to ensure the appropriate response to a given set of environmental conditions.  In S. cerevisiae, the PKA and Tor pathways are central elements of this control.  The Tor protein kinases regulate growth in all eukaryotes and represent important targets for cancer therapy.  We are interested in the interplay between these signaling pathways and have been examining how changes in one pathway influence the activity of the other.  Our studies to date indicate that these pathways function independently to control growth; neither pathway appears to be under the auspices of the other.  However, there does appear to be crosstalk occurring between these pathways and our present work is aimed at understanding the role that this information flow plays in controlling growth.