Faculty Bibliography

Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2)O(2)), act as intracellular second messengers in many signaling pathways. Protein-tyrosine phosphatases (PTPs) are now believed to be important targets of ROS. PTPs contain a conserved catalytic cysteine with an unusually low pK(a). This property allows PTPs to execute nucleophilic attack on substrate phosphotyrosyl residues, but also renders them highly susceptible to oxidation. Reversible oxidation, which inactivates PTPs, is emerging as an important cellular regulatory mechanism and might contribute to human diseases, including cancer. Given their potential toxicity, it seems likely that ROS generation is highly controlled within cells to restrict oxidation to those PTPs that must be inactivated for signaling to proceed. Thus, identifying ROS-inactivated PTPs could be tantamount to finding the PTP(s) that critically regulate a specific signaling pathway. This article provides an overview of the methods currently available to identify and quantify PTP oxidation and outlines future challenges in redox signaling.

Recently, loss-of-function mutations in PTPN11 were linked to the cartilage tumor syndrome metachondromatosis (MC), a rare inherited disorder featuring osteochondromas, endochondromas and skeletal deformation. However, the underlying molecular and cellular mechanism for MC remained incompletely understood. By studying the role of the Src homology-2 domain-containing protein tyrosine phosphatase Shp2 (encoded by mouse Ptpn11) in cathepsin K-expressing cells, we identified a novel cell population in the perichondrial groove of Ranvier. In the absence of Shp2, these cells exhibit elevated Indian hedgehog (Ihh) signaling, proliferate excessively and cause ectopic cartilage formation and tumors. Our findings establish a critical role for a protein-tyrosine phosphatase (PTP) family member, in addition to the well-known roles of receptor tyrosine kinases (RTKs), in cartilage development and homeostasis. However, whether Shp2 deficiency in other epiphyseal chondroid cells and whether signaling pathways in addition to the IHH/Parathyroid Hormone-related Peptide (PTHrP) axis attribute to the formation of enchondromas and osteochondromas remains elusive. Understanding how chondrogenic events are regulated by SHP2 could aid in the development of novel therapeutic approaches to prevent and treat cartilage diseases, such as MC and osteoarthritis (OA).

Ovarian cancer is the leading cause of morbidity/mortality from gynecologic malignancy. Early detection of disease is difficult due to the propensity for ovarian cancer to disseminate throughout the peritoneum. Currently, there is no single accurate test to detect primary or recurrent ovarian cancer. We report a novel clinical strategy using PPF: a multimodal, PET and optical, folate receptor (FR)-targeted agent for ovarian cancer imaging. The capabilities of PPF were evaluated in primary human ovarian cancer cells, in vivo xenografts derived from primary cells and ex vivo patient omemtum, as the heterogeneity and phenotype displayed by patients is retained. Primary cells uptake PPF in a FR-dependent manner demonstrating approximately a 5- to 25-fold increase in fluorescence. By both PET and fluorescence imaging, PPF specifically delineated FR-positive, ovarian cancer xenografts, with similar tumor-to-background ratios of 8.91+/-0.91 and 7.94+/-3.94, and micro-metastatic studding (<1mm), which demonstrated a 3.5-fold increase in PPF uptake over adjacent normal tissue. Ex vivo patient omentum demonstrated selective uptake of PFF by tumor deposits. The ability of PPF to identify metastatic deposits <1mm could facilitate more complete debulking (currently, optimal debulking is <10mm residual tumor), by providing a more sensitive imaging strategy improving treatment planning, response assessment and residual/recurrent disease detection. Therefore, PPF is a novel clinical imaging strategy that could substantially improve the prognosis of patients with ovarian cancer by allowing pre-, post- and intra-operative tumor monitoring, detection and possibly treatment throughout all stages of therapy and tumor progression.

INTRODUCTION: The mouse mammary gland provides a powerful model system for studying processes involved in epithelial tissue development. Although markers that enrich for mammary stem cells and progenitors have been identified, our understanding of the mammary developmental hierarchy remains incomplete. METHODS: We used the MMTV promoter linked to the reverse tetracycline transactivator to induce H2BGFP expression in the mouse mammary gland. Mammary epithelial cells (MECs) from virgin mice were sorted by flow cytometry for expression of the mammary stem cell/progenitor markers CD24 and CD29, and H2BGFP. Sorted populations were analyzed for in vivo repopulation ability, expression of mammary lineage markers, and differential gene expression. RESULTS: The reconstituting activity of CD24(+)/CD29(+) cells in cleared fat pad transplantation assays was not distinguished in GFP(+) compared to GFP(-) subpopulations. However, within the CD24(+)/CD29(lo) luminal progenitor-enriched population, H2BGFP(+), but not H2BGFP(-), MECs formed mammary structures in transplantation assays; moreover, this activity was dramatically enhanced in pregnant recipients. These outgrowths contained luminal and myoepithelial mammary lineages and produced milk, but lacked the capacity for serial transplantation. Transcriptional microarray analysis revealed that H2BGFP(+)/CD24(+)/CD29(lo) MECs are distinct from H2BGFP(-)/CD24(+)/CD29(lo) MECs and enriched for gene expression signatures with both the stem cell (CD24(+)/CD29(+)) and luminal progenitor (CD24(+)/CD29(lo)/CD61(+)) compartments. CONCLUSIONS: We have identified a population of MECs containing pregnancy-activated multipotent progenitors that are present in the virgin mammary gland and contribute to the expansion of the mammary gland during pregnancy.

Protein functions are largely affected by their conformations. This is exemplified in proteins containing modular domains. However, the evolutionary dynamics that define and adapt the conformation of such modular proteins remain elusive. Here we show that cis-interactions between the C-terminal phosphotyrosines and SH2 domain within the protein tyrosine phosphatase Shp2 can be tuned by an adaptor protein, Grb2. The competitiveness of two phosphotyrosines, namely pY542 and pY580, for cis-interaction with the same SH2 domain is governed by an antagonistic combination of contextual amino acid sequence and position of the phosphotyrosines. Specifically, pY580 with the combination of a favourable position and an adverse sequence has an overall advantage over pY542. Swapping the sequences of pY542 and pY580 results in one dominant form of cis-interaction and subsequently inhibits the trans-regulation by Grb2. Thus, the antagonistic combination of sequence and position may serve as a basic design principle for proteins with tunable conformations.

OBJECTIVE: The receptor-like protein-tyrosine phosphatase (PTP) CD148 and the nontransmembrane PTP1-B have been shown to be net positive regulators of Src family kinases in platelets. In the present study, we compared the relative contributions of these PTPs in platelet activation by the major glycoprotein, glycoprotein VI, alpha(IIb)beta(3), and C-type lectin-like receptor 2 (CLEC-2). METHODS AND RESULTS: PTP-1B-deficient mouse platelets responded normally to the glycoprotein VI-specific agonist collagen-related peptide and antibody-mediated CLEC-2 activation. However, they exhibited a marginal reduction in alpha(IIb)beta(3)-mediated Src family kinase activation and tyrosine phosphorylation. In contrast, CD148-deficient platelets exhibited a dramatic reduction in activation by glycoprotein VI and alpha(IIb)beta(3) and a marginal reduction in response to activation by CLEC-2, which was further enhanced in the absence of PTP-1B. These defects were associated with reduced activation of Src family kinase and spleen tyrosine kinase, suggesting a causal relationship. Under arteriolar flow conditions, there was defective aggregate formation in the absence of PTP-1B and, to a greater extent, CD148 and a severe abrogation of both adhesion and aggregation in the absence of both PTPs. CONCLUSIONS: Findings from this study demonstrate that CD148 plays a dominant role in activating Src family kinases in platelets relative to PTP-1B. Both PTPs are required for optimal platelet activation and aggregate formation under high arterial shear rates.

Macrophages migrate to sites of insult during normal inflammatory responses. Integrins guide such migration, but the transmission of signals from integrins into the requisite cytoskeletal changes is poorly understood. We have discovered that the hematopoietic adaptor protein Skap2 is necessary for macrophage migration, chemotaxis, global actin reorganization and local actin reorganization upon integrin engagement. Binding of phosphatidylinositol [3,4,5]-triphosphate to the Skap2 pleckstrin-homology (PH) domain, which relieves its conformational auto-inhibition, is critical for this integrin-driven cytoskeletal response. Skap2 enables integrin-induced tyrosyl phosphorylation of Src-family kinases (SFKs), Adap, and Sirpalpha, establishing their roles as signaling partners in this process. Furthermore, macrophages lacking functional Sirpalpha unexpectedly have impaired local integrin-induced responses identical to those of Skap2(-/-) macrophages, and Skap2 requires Sirpalpha for its recruitment to engaged integrins and for coordinating downstream actin rearrangement. By revealing the positive-regulatory role of Sirpalpha in a Skap2-mediated mechanism connecting integrin engagement with cytoskeletal rearrangement, these data demonstrate that Sirpalpha is not exclusively immunoinhibitory, and illuminate previously unexplained observations implicating Skap2 and Sirpalpha in mouse models of inflammatory disease.

The pentaspan membrane glycoprotein CD133 marks lineage-specific cancer progenitor cells and is associated with poor prognosis in a number of tumor types. Despite its utility as a cancer progenitor cell marker, CD133 protein regulation and molecular function remain poorly understood. We find that the deacetylase HDAC6 physically associates with CD133 to negatively regulate CD133 trafficking down the endosomal-lysosomal pathway for degradation. We further demonstrate that CD133, HDAC6, and the central molecule of the canonical Wnt signaling pathway, beta-catenin, can physically associate as a ternary complex. This association stabilizes beta-catenin via HDAC6 deacetylase activity, which leads to activation of beta-catenin signaling targets. Downregulation of either CD133 or HDAC6 results in increased beta-catenin acetylation and degradation, which correlates with decreased proliferation in vitro and tumor xenograft growth in vivo. Given that CD133 marks progenitor cells in a wide range of cancers, targeting CD133 may be a means to treat multiple cancer types.

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 ( approximately 3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1(D486N). Raf1(D486N/+) (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1(D486N)-expressing cells compared with controls. RAF1(D486N), as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypophosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased transepithelial resistance, and lateral domain shortening. Conversely, GAB1 overexpression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multilumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a negative regulator of epithelial cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane.