A mediator of Rho-dependent invasion moonlights as a methionine salvage enzyme.

RhoA controls changes in cell morphology and invasion associated with cancer phenotypes. Cell lines derived from melanoma tumors at varying stages revealed that RhoA is selectively activated in cells of metastatic origin. We describe a functional proteomics strategy to identify proteins regulated by RhoA and report a previously uncharacterized human protein, named "mediator of RhoA-dependent invasion (MRDI)," that is induced in metastatic cells by constitutive RhoA activation and promotes cell invasion. In human melanomas, MRDI localization correlated with stage, showing nuclear localization in nevi and early stage tumors and cytoplasmic localization with plasma membrane accentuation in late stage tumors. Consistent with its role in promoting cell invasion, MRDI localized to cell protrusions and leading edge membranes in cultured cells and was required for cell motility, tyrosine phosphorylation of focal adhesion kinase, and modulation of actin stress fibers. Unexpectedly MRDI had enzymatic function as an isomerase that converts the S-adenosylmethionine catabolite 5-methylribose 1-phosphate into 5-methylribulose 1-phosphate. The enzymatic function of MRDI was required for methionine salvage from S-adenosylmethionine but distinct from its function in cell invasion. Thus, mechanisms used by signal transduction pathways to control cell movement have evolved from proteins with ancient function in amino acid metabolism.

Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine.

Alpha-1 acid glycoprotein (AGP, orosomucoid), a major acute phase protein in plasma, displays potent cytoprotective and anti-inflammatory activities whose molecular mechanisms are largely unknown. Because AGP binds various exogenous drugs, we have searched for endogenous ligands for AGP. We found that AGP binds lysophospholipids in a manner discernible from albumin in several ways. First, mass spectrometric analyses showed that AGP isolated from plasma and serum contained lysophosphatidylcholine (LPC) enriched in mono and polysaturated acyl chains, whereas albumin contained mostly saturated LPC. Second, AGP bound LPC in a 1:1 molar ratio and with a higher affinity than free fatty acids, whereas albumin bound LPC in a 3:1 ratio but with a lower affinity than that of free fatty acids. Consequently, free fatty acids displaced LPC more avidly from albumin than from AGP. Competitive ligand displacement indicated the highest affinity for AGP to LPC20:4, 18:3, 18:1, and 16:0 (150-180 nM), lysophosphatidylserine (Kd 190 nM), and platelet activating factor (PAF) (Kd 235 nM). The high affinity of AGP to LPC in equilibrium was verified by stopped-flow kinetics, which implicated slow dissociation after fast initial binding, being consistent with an induced-fit mechanism. AGP also bound pyrene-labeled phospholipids directly from vesicles and more efficiently than albumin. AGP prevented LPC-induced priming and PAF-induced activation of human granulocytes, thus indicating scavenging of the cellular effects of the lipid ligands. The results suggest that AGP complements albumin as a lysophospholipid scavenging protein, particularly in inflammatory conditions when the capacity of albumin to sequester LPC becomes impaired.

Functional proteomics identifies protein-tyrosine phosphatase 1B as a target of RhoA signaling.

Rho GTPases are signal transduction effectors that control cell motility, cell attachment, and cell shape by the control of actin polymerization and tyrosine phosphorylation. To identify cellular targets regulated by Rho GTPases, we screened global protein responses to Rac1, Cdc42, and RhoA activation by two-dimensional gel electrophoresis and mass spectrometry. A total of 22 targets were identified of which 19 had never been previously linked to Rho GTPase pathways, providing novel insight into pathway function. One novel target of RhoA was protein-tyrosine phosphatase 1B (PTP1B), which catalyzes dephosphorylation of key signaling molecules in response to activation of diverse pathways. Subsequent analysis demonstrated that RhoA enhances post-translational modification of PTP1B, inactivates phosphotyrosine phosphatase activity, and up-regulates tyrosine phosphorylation of p130Cas, a key mediator of focal adhesion turnover and cell migration. Thus, protein profiling reveals a novel role for PTP1B as a mediator of RhoA-dependent phosphorylation of p130Cas.

Two-dimensional gel electrophoresis for the identification of signaling targets.

Two-dimensional electrophoresis (2-DE) is a powerful technique to differentially display patterns of protein expression and posttranslational modifications, providing a good strategy to monitor molecular responses induced by the activation or inactivation of specific signaling pathways. In this chapter, optimized protocols for 2-DE using extracts from tissue culture are provided. Protocols for in-gel digestion of gel-resolved proteins, which allow protein identification by mass spectrometry are also discussed.

Functional proteomic analysis of melanoma progression.

Functional proteomics provides a powerful approach to screen for alterations in protein expression and posttranslational modifications under conditions of human disease. In this study, we use protein screening to examine markers of melanoma progression, by profiling melanocyte versus melanoma cell lines using two-dimensional electrophoresis and mass spectrometry. Eight candidate markers were identified as differentially regulated in transformed cells. In particular, hepatoma-derived growth factor (HDGF) and nucleophosmin B23 were strongly correlated with melanoma. Nucleophosmin B23 is a nucleolar and centrosome-associated protein, which has been implicated as a target for cyclin E/cyclin-dependent kinase 2 (CDK2) in modulating centrosome duplication and cell cycle control. Western blotting of one-dimensional and two-dimensional gels showed that the form of nucleophosmin B23 that is up-regulated in melanoma represents a posttranslationally modified form, most likely reflecting enhanced phosphorylation in the tumor-derived cells. In contrast, Western analysis of HDGF demonstrated increased expression of all forms in melanoma cell lines compared with melanocytes. Immunohistochemical analysis of human tissue biopsies showed strong expression of HDGF in early and late stage melanomas and low expression in melanocytes and nontumorigenic nevi. Interestingly, biopsies of nevi showed a graded effect in which HDGF immunoreactivity was reduced in nevoid nests penetrating deep into the dermis compared with nests at the epidermal-dermal junction, suggesting that HDGF expression in nevi is dependent on epidermal cell interactions. In contrast, biopsies of melanoma showed strong expression of HDGF throughout the tumor, including cells located deeply within dermis. Thus, expression of this antigen likely reports a reduced dependence of protein expression on epidermal interactions.