Section: Research

Alonzo Ross, Ph.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Biochemistry and Molecular Pharmacology

Other Affiliation(s):
   Interdisciplinary Graduate Program
   Program in Neuroscience

PTEN phosphatase and tumor suppressor, CNS Stem Cells and neural tumors.

We have 5 current projects, all related to the PTEN tumor suppressor. First, even though phosphorylation of phosphatidylinositols by phosphoinositide 3-kinase has an important and pervasive role in the nervous system, little is known about the phosphatases that reverse this reaction. Recently, such a phosphatase, PTEN, was cloned as a tumor suppressor for gliomas. We now know that PTEN is a tumor suppressor for many tumor types and is a phosphatidylinositol phosphatase specific for the 3-position of the inositol ring. PTEN is expressed in most, if not all, neurons and is localized in the nucleus and cytoplasm. PTEN is induced during neuronal differentiation. Studies are in progress to determine which downstream pathways are regulated by PTEN, by which mechanisms PTEN activity is regulated, which stimuli regulate PTEN activity and why a molecule that inhibits several survival pathways is induced during neurogenesis.

Figure 1. Balance between phosphatidylinositol 4,5 bisphosphate and phosphatidylinositol 3,4,5 trisphosphate is determined by relative activities of phosphatidylinositol 3-kinase and PTEN phosphatase.

Second, we are examining the role of nuclear PTEN. Despite the potential importance of nuclear PTEN in tumor development, no nuclear localization signal (NLS) has been identified. In addition, no clear physiological effects of nuclear PTEN are known. We have recently prepared mutant PTENs with decreased or increased nuclear localization that can be used to define the functions of nuclear PTEN. We have identified a candidate NLS sequence and have determined that both nuclear and cytoplasmic PTEN can induce apoptosis. We currently are identifying the PTEN NLS and analyzing the mechanism of cell death induced by nuclear PTEN.

Figure 2. Micrograph showing that exogenously expressed green fluorescent protein PTEN is present in both the nucleus and cytoplasm of HeLa cells.

Figure 3. Magnetic resonance image of patient's brain bearing a large glioma brain tumor.

Third, we are developing mouse models for PTEN loss and development of brain cancer. Many changes occur during the development of brain tumors, including loss of PTEN. We can now grow cultures of PTEN -/- CNS stem cells and then infect with oncogene-bearing retroviruses. We currently are examining cells that have lost PTEN and express a mutated epidermal growth factor receptor, EGFRvIII, which is constitutively activated and frequently expressed in human tumors. These PTEN -/- EGFRvIII+ cells have some characteristics of transformed cells but not others. We have also characterized gene expression in PTEN -/- stem cells and have identified a new marker induced by PTEN loss. With Tom Smith in the Pathology Department and Scott Litofsky from Neuro-surgery, we are extending these results to human tumors.

Figure 4. Electron micrograph showing yeast cell. Recently we have identified a yeast PTEN protein.

Fourth, we have recently found a close homologue of PTEN in yeast. This was an unexpected finding since yeast lack the Class I phosphoinositide 3-kinases that generate PI(3,4,5)P₃ in higher eukaryotes. Indeed, PI(3,4,5)P₃ has not been detected in yeast. Surprisingly, we found that upon deletion of the yeast PTEN, PI(3,4,5)P₃ became detectable at levels comparable to those found in mammalian cells, indicating that a pathway exists for synthesis of this lipid and that the yeast PTEN, like mammalian PTEN, acts to suppress PI(3,4,5)P₃ levels. We now are carrying out genetic screens to elucidate the PI(3,4,5)P₃ pathway in yeast.

Activation of PTEN by PI(4,5)P₂. Data were fit to the equation
V = V_act[PI(4,5)P₂]/(K_act + [PI(4,5)P₂]).

Figure 5. Fit of kinetic data for activation of PTEN by phosphatidylinositol 4,5 bisphosphate.

Fifth, we are examining the regulation of PTEN activity at the protein level. In recent experiments, we measured reaction rates for varying concentrations of monodisperse (i.e., not in a vesicle or micelle) PI(3,4,5)P₃. The kinetic curves did not follow the typical Michaelis-Menten form, especially at higher PI(3,4,5)P₃ levels. The kinetic curves were sigmoidal, indicating that the enzymatic activity increases as the reaction progresses. One possible explanation is that the PTEN product, PI(4,5)P₂, is a positive regulator of PTEN activity. We measured PTEN activity as a function of PI(4,5)P₂ concentration and found that PI(4,5)P₂ activated PTEN with a K_act = 20 micromolar. This regulation is specific. For example, PI(3,4)P₂ and PI(3,5)P₂ do not activate PTEN. Based on these data, we propose that PI(4,5)P₂ binds to a site distinct from the phosphatase active site, induces an allosteric conformational change, and, thereby, activates PTEN, leading to a positive feedback loop for PTEN activity. This model predicts that PTEN would be preferentially activated at the PI(4,5)P₂-bearing plasma membrane or at PI(4,5)P₂ -rich membrane domains.

Office: LRB-819, Lab 870 I-K
Phone: 508-856-8016
E-mail: Alonzo.Ross@umassmed.edu
Keywords: Neurobiology, Cancer Biology, Stem Cell Biology, Biochemistry, Oncogenes/tumor suppressors

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