Jonathan Chernoff, MD, PhD
Office Phone: 215-728-5319
Lab Phone: 215-728-5320
Substrates of p21-Activated Kinases
p21-activated kinases (Paks) are effectors for the Rho GTPases Cdc42 and Rac, and are central players in cell proliferation, survival, and motility. While a number of key substrates for Paks have been identified, no systematic survey has ever been undertaken to uncover the targets of this protein kinase. We are currently using two unbiased screening methods to identify such substrates in mast cells and breast epithelial cells.
Understanding Paks involvement in neurofibromatosis type 2 (NF2), an autosomal dominant disorderHoi Yee (Betty) Chow
This project is aimed at understanding the mechanism by which Paks are involved in neurofibromatosis type 2 (NF2), an autosomal dominant disorder. Paks are downstream effector proteins of Rac1/Cdc42 that contribute to cytoskeletal reorganization, which is often associated with cellular transformation. Also, Pak1 can phosphorylate merlin, the gene product of the NF2 gene, to inactivate its tumor suppressor function. The goal of this research is to examine the signaling pathways operating between Paks and merlin, and understand how they are regulated. The approach for this research uses specific polypeptide or small molecule inhibitors to determine whether loss of Paks function affects disease progression and the biological behavior of NF2-deficient animals. The successful completion of these studies will increase our basic understanding of signal transduction and should determine whether Pak is a useful target in the treatment of this devastating disorder. It may even provide a strategy to develop a new compound for further therapeutics.Top
Genetic and Biochemical Analysis of Pak function
We have made constitutive and conditional knock out mouse models for the two most hightly expressed Pak isoforms: Pak1 and Pak2. We are using these mice, in conjunction with disease models for Ras-driven skin cancer, NF1, NF2, and HER2-driven breast cancer to assess the role of Paks in these malignancies.
Uncovering new Pak2 substrates and their signaling pathwaysMaria Radu
The Pak family is an important player in regulation of cell motility, actin reorganization, gene transcription and apoptosis. Pak I family members share at least 93% homology in their kinase domain, suggesting identical or overlapping functions. However, knock out mice for the three individual members, Pak1, Pak2 and Pak3 have very distinct phenotypes. Pak1 and Pak3 knock out mice are viable and fertile and have mild changes in their phenotype. Pak2 knock out mice are embryonic lethal which hints towards the fact that Pak2 has distinct functions in development. A number of Pak2 substrates are well known and characterized, but a multitude of cellular events mediated by Pak2 are still a mystery. We are using a biochemical based approach to identify Pak2 specific targets and their role in mediating specific cellular events.Top
Examine the role phosphorylation events have in cell motility and survival
p21-Activated Kinase 1 (Pak1) is a serine-threonine kinase functioning in cytoskeleton rearrangement, cell motility and the activation of transcriptional pathways. Studies indicate increased Pak1 protein and activity levels are linked to breast, gastric, ovarian and brain cancers. Pak1 is a unique kinase, phosphorylating both cytoplasmic and nuclear proteins. Specifically, phosphorylation of such nuclear targets as the estrogen receptor alpha, SHARP, and Snai1, has been shown to promote cancerous phenotypes, suggesting distinct cytoplasmic and nuclear roles of Pak1 which lead to metastasis. We seek to elucidate the Pak1 nuclear import pathway through abrogating key Pak1-protein interactions important for kinase activation, assessing nuclear accumulation using immunofluorescence, and examining the ability for Pak1 to interact with other known binding partners. We look to distinguish between these compartmental roles by selectively studying Pak1 target proteins in cytoplasmic and nuclear cell fractions, and then examining the role these phosphorylation events have in cell motility and survival. Finally, using the zebrafish in vivo model system, we will study the effect known Pak1-protein interactions and precise Pak1 signaling pathways have on organism development and survival. Results obtained will provide insight into potential regulators of Pak1 activity, giving rise to the development of novel cancer therapeutics.
The role of Pak1 in ErbB2/Neu signaling
The activation of receptor tyrosine kinases, particularly ErbB2, plays an important role in the genesis of breast cancer. ErbB2 kinase activity promotes Ras-mediated stimulation of a downstream kinase cascade, which includes the Raf-1/MEK/extracellular-signal regulated kinase 1/2 (ERK1/2) pathway leading to tumor cell growth and migration. Moreover, it is well documented that signaling through the Ras-ERK1/2 pathway can be influenced by p21-activated kinase 1 (Pak1), an effector of the Rho family GTPases Rac and Cdc42. Expression of constitutively active Rac and Cdc42 can synergize with Raf to promote activation of ERK1/2 through mechanisms involving Pak1 phosphorylation of MEK1 and Raf-1. Surprisingly, the information of the contribution of Rho GTPases to ErbB2 mediated signaling is very limited. We are interested in clarifying the molecular mechanism(s) by which the Rac-Pak pathway contributes to ErbB2/Neu mediated transformation by using a three dimensional cell culture system as well as a mouse model of breast cancer.
Design of genetically-encodable, FRET-based biosensors for group I Paks
Protein kinases are crucial components of intracellular signaling pathways which transmit signals by phosphorylation of downstream targets, altering their function. Traditional methods to measure Pak signaling, by Western blotting or immunostaining for phosphorylated, active Pak, have provided valuable insight into Pak function. However, these methods present a static snapshot of cellular events; they do not allow for the dynamic examination of Pak activity with fine spatial resolution. The goal for this research is to create a genetically encoded FRET-based sensor of Pak activity that selectively reports group I Paks signaling in living cells.Top
Regulation of Protein Phosphatases by Sumoylation
We recently discovered that protein tyrosine phosphatase (PTP)-1B, a major regulator of insulin signaling, is itself regulated by sumoylation. The attachment of SUMO groups to proteins has been recognized as a means to modify location and protein/protein interactions, but there are few reports of sumoylation affecting catalytic activity of an enzyme. In the case of PTP-1B, sumoylation of one or more lysine residues strongly inhibits the catalytic activity of this phosphatase. We also found that sumoylation of PTP-1B is itself transiently stimulated by insulin. These findings suggest a new model for regulating signaling by the insulin receptor and perhaps other receptor tyrosine kinases.
PTP1B is a widely expressed tyrosine-specific protein phosphatase that functions as a key regulator of insulin signaling. Overexpression of PTP1B impairs insulin signals, whereas loss of PTP1B is associated with increase sensitivity to insulin. For these reasons, it is vital to understand how PTP1B is regulated. We have recently established that PTP1B associates with a SUMO E3 ligase, PIAS1, thereby promoting sumoylation of PTP1B. Sumoylation is a post-translational modification, and sumoylation of PTP1B sharply reduces its catalytic activity, resulting in loss of its ability to dephosphorylate and inactivate the insulin receptor. Surprisingly, the ER resident PTP1B is partially co-localized with RFP-SUMO and GFP-lamin at the nuclear envelope, suggesting a new role for sumoylated PTP1B at the nuclear membrane. To determine the cellular function of PTP1B at the INM, we ask if PTP1B binds to INM proteins, such as Lamin A/C and Emerin. Mutation of Lamin and Emerin have been closely linked to the early aging diseases such as Progeria and Muscular Dystrophy. Using substrate trapping mutant of PTP1B, Lamin and Emerin co-immunoprecipitated with PTP1B, suggesting its interaction. Although no genetic mutation has been reported on the tyrosine residue of Lamin or Emerin, the effect of sumoylated PTP1B on tyrosine phosphorylation of Lamin or Emerin will be further investigated.
Study the role of PTP1B in ErbB2/Neu signaling
Protein Tyrosine Phosphatase 1B (PTP1B) has long been known to play a major role in inhibiting signaling from the insulin and leptin receptors. As such, this enzyme has been considered to be a suppressor of growth. However, recently, this enzyme has been found to play a positive role in ErbB2/Neu signaling in mouse models of breast cancer. This surprising finding suggests that PTP1B activates key signaling elements downstream of ErbB2/Neu, and that inhibiting PTP1B might be beneficial in certain forms of breast cancer. In this capactiy, we look to:
a) Identify the key substrates of PTP1B that mediate its positive effects on ErbB2/Neu signaling in a three-dimensional cell culture system.Top
b) Analyze the role of PTP1B in ErbB2/Neu mediated carcinogenesis in vivo.
Role of PTP1B in Insulin Signaling
PTP1B contains two binding motifs that direct it to its substrates: a dityrosine motif in the N terminus that is required for stable association with the insulin receptor; and a proline-rich region in the C-terminus that directs binding to SH3-containing proteins such as Src. We have recently creating mouse knock-in models bearing mutants of PTP1B in these two domains. A current project is to study these PTP1B knock-in mice for defects in insulin and HER2 signaling.
Define the targets of PTP1B that contribute to HER2 signaling in human breast epithelial cells.
PTP1B plays a paradoxical role in breast cancer. Rather than acting as a growth suppressor, it acts in concert with HER2 as an oncogene. At present, we do not know how PTP1B contributes to HER2 signaling or breast cancer. We are using a quantitative proteomics method, SILAC, in combination with substrate trapping, to define the targets of PTP1B that contribute to HER2 signaling in human breast epithelial cells.Top
Rho GTPases in Formation of Breast Acini in 3D Culture
Rho GTPases and their effectors play a major role in regulating the actin and tubulin cytoskeletons. We are using three dimensional cell culture techniques to study the effects and molecular mechanisms of Rho GTPases on acinar development of breast epithelial cells.Top
Analysis of Protein Phosphorylation in Stem Cell Pluripotenc
While the core transcriptional machinery that is responsible for maintaining pluripotency in mouse embryonic stem cells has been identified, little is known of the signaling pathways upstream of these nuclear factors. We are currently engaged in a kinome/phosphatome-wide siRNA screen for enzymes that are required to maintain pluripotency, as well as enzymes that inhibit this process.Top