Endofin is required for HD-PTP and ESCRT-0 interdependent endosomal sorting of ubiquitinated transmembrane cargoes.

Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.

Haploinsufficiency of the ESCRT Component HD-PTP Predisposes to Cancer.

Endosomal sorting complexes required for transport (ESCRT) drive cell surface receptor degradation resulting in attenuation of oncogenic signaling and pointing to a tumor suppressor function. Here, we show that loss of function of an ESCRT protein (HD-PTP encoded by the PTPN23 gene, located on the tumor suppressor gene cluster 3p21.3) drives tumorigenesis in vivo. Indeed, Ptpn23(+/-) loss predisposes mice to sporadic lung adenoma, B cell lymphoma, and promotes Myc-driven lymphoma onset, dissemination, and aggressiveness. Ptpn23(+/-)-derived tumors exhibit an unaltered remaining allele and maintain 50% of HD-PTP expression. Consistent with the role of HD-PTP in attenuation of integrin recycling, cell migration, and invasion, hemizygous Ptpn23(+/-) loss increases integrin ß1-dependent B cell lymphoma survival and dissemination. Finally, we reveal frequent PTPN23 deletion and downregulation in human tumors that correlates with poor survival. Altogether, we establish HD-PTP/PTPN23 as a prominent haploinsufficient tumor suppressor gene preventing tumor progression through control of integrin trafficking.

Interplay of Endosomal pH and Ligand Occupancy in Integrin α5ß1 Ubiquitination, Endocytic Sorting, and Cell Migration.

Membrane trafficking of integrins plays a pivotal role in cell proliferation and migration. How endocytosed integrins are targeted either for recycling or lysosomal delivery is not fully understood. Here, we show that fibronectin (FN) binding to α5ß1 integrin triggers ubiquitination and internalization of the receptor complex. Acidification facilitates FN dissociation from integrin α5ß1 in vitro and in early endosomes, promoting receptor complex deubiquitination by the USP9x and recycling to the cell surface. Depending on residual ligand occupancy of receptors, some α5ß1 integrins remain ubiquitinated and are captured by ESCRT-0/I, containing histidine domain-containing protein tyrosine phosphatase (HD-PTP) and ubiquitin-associated protein 1 (UBAP1), and are directed for lysosomal proteolysis, limiting receptor downstream signaling and cell migration. Thus, HD-PTP or UBAP1 depletion confers a pro-invasive phenotype. Thus, pH-dependent FN-integrin dissociation and deubiquitination of the activated integrin α5ß1 are required for receptor resensitization and cell migration, representing potential targets to modulate tumor invasiveness.

Folliculin regulates ampk-dependent autophagy and metabolic stress survival.

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation.

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Pseudophosphatases: methods of analysis and physiological functions.

Protein tyrosine phosphatases (PTPs) are key enzymes in the regulation of cellular homeostasis and signaling pathways. Strikingly, not all PTPs bear enzymatic activity. A considerable fraction of PTPs are enzymatically inactive and are known as pseudophosphatases. Despite the lack of activity they execute pivotal roles in development, cell biology and human disease. The present review is focused on the methods used to identify pseudophosphatases, their targets, and physiological roles. We present a strategy for detailed enzymatic analysis of inactive PTPs, regulation of inactive PTP domains and identification of binding partners. Furthermore, we provide a detailed overview of human pseudophosphatases and discuss their regulation of cellular processes and functions in human pathologies.

Expression analysis and essential role of the putative tyrosine phosphatase His-domain-containing protein tyrosine phosphatase (HD-PTP).

The putative tyrosine phosphatase HD-PTP, encoded by the protein-tyrosine-phosphatase-n23 (Ptpn23) gene, has been described as a tumor suppressor candidate gene. However, its physiological roles and detailed expression profiles are poorly defined. To investigate HD-PTP functions, we generated a mouse model in which the Ptpn23 locus was disrupted by an in-frame insertion of a beta-galactosidase-neomycin-phosphotransferase II (beta-geo) cassette. This insertion leads to the expression of a catalytically inactive truncated protein preserving only the uncharacterized N-terminal BRO1-like domain in fusion with beta-geo under the control of the endogenous promoter. Here we report that homozygous gene deletion is lethal around embryonic day 9.5, suggesting that Ptpn23 is an essential requirement for early stages of embryonic development. Taking advantage of the beta-galactosidase insertion into the Ptpn23 locus, we define the precise Ptpn23 expression pattern by performing X-gal staining at different stages of mouse development. Our results show that Ptpn23 is expressed early during mouse development and that its expression is maintained in adult tissues, markedly in the epithelial cells of many organs.

HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain.

BACKGROUND: The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP). To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported. METHODOLOGY AND RESULTS: Here we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status. CONCLUSION: In summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.