The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum

AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA⁻ cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA⁻ cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.Physiological processes that define and maintain the sizes of tissues are poorly understood. Although a number of characterized gene products negatively regulate the sizes of tissues (21, 23), the mechanism by which the activities of such gene products are controlled is unclear. One potential mechanism for tissue size regulation consists of tissue-specific autocrine signals that inhibit proliferation in a concentration-dependent manner (18). Since the extracellular concentration of such factors increases as a function of cell density and/or cell number, the proliferation-inhibiting function of these factors can limit tissue size. Considerable evidence for such factors has been reported. For instance, full hepatectomy in one of two rats with conjoined circulatory systems stimulated proliferation in the intact liver of the conjoined rat, suggesting the existence of a systemic factor produced by the liver that inhibits the proliferation of hepatocytes (16). However, only a small number of factors with analogous functional roles, such as myostatin, which regulates skeletal muscle size (30), and Gdf11, which negatively regulates neurogenesis in the olfactory epithelium (38), have been identified. The mechanisms by which such signals inhibit proliferation are not well understood. As such autocrine signals may serve to limit tumor growth (14, 20), elucidation of the identities of such factors and their associated signal transduction pathways may yield novel cancer therapies.We have identified two such autocrine proliferation-repressing signals in the social amoeba Dictyostelium discoideum, a genetically and biochemically tractable model organism. The proteins AprA and CfaD are secreted by Dictyostelium and inhibit the proliferation of Dictyostelium cells in a concentration-dependent manner (4, 12). Cells in which the genes encoding either AprA or CfaD have been disrupted by homologous recombination proliferate rapidly, and cells overexpressing AprA or CfaD proliferate slowly (4, 11). Adding recombinant AprA (rAprA) or recombinant CfaD (rCfaD) to cells slows proliferation, demonstrating that these proteins function as extracellular signals (4, 12). In addition to exhibiting rapid proliferation, aprA⁻ and cfaD⁻ cells exhibit a multinucleate phenotype, strongly suggesting that AprA and CfaD are negative regulators of mitosis (4, 11). aprA⁻ cells are insensitive to the proliferation-inhibiting effects of CfaD (12), and cfaD⁻ cells are insensitive to AprA (4), indicating the necessity of both genes for proliferation inhibition and suggesting a common proliferation-inhibiting mechanism. The G protein complex subunits Gα8, Gα9, and Gβ are necessary for proliferation inhibition by AprA, and the addition of recombinant AprA to purified cell membranes increases binding of GTP to wild-type and gα9⁻ cell membranes but not gα8⁻ or gβ⁻ membranes, indicating that AprA activates a proliferation-inhibiting signal transduction pathway of which Gα8 and Gβ are components (5). The signal transduction pathway downstream of Gα8 and the associated mechanism of proliferation inhibition are unknown.Although the selective forces that have maintained functional autocrine proliferation inhibitors in proliferating Dictyostelium cells are unclear, AprA and CfaD may provide an advantage during the multicellular portion of the Dictyostelium life cycle. Upon starvation, Dictyostelium cells secrete pulses of the chemoattractant cyclic AMP, leading to cells streaming toward aggregation centers (15, 27). This process causes the formation of multicellular groups regulated in size by a secreted protein complex that stimulates stream breakup (9, 10). These groups develop into multicellular fruiting body structures composed of a mass of stress-resistant spores supported by an approximately 1-mm-high stalk (24). While the stalk cells inevitably die in an act of apparent altruism (31), the presence of nutrients stimulates spore germination and a continuation of proliferation (13). Following development, aprA⁻ and cfaD⁻ cells form fewer viable spores than the wild type (4, 11), suggesting that AprA and CfaD increase the fitness of Dictyostelium during development.Like aprA⁻ and cfaD⁻ cells, Dictyostelium cells lacking the ROCO family kinase QkgA have an abnormally rapid proliferation (1). The ROCO protein family is widely conserved and is defined by the presence of a Ras of complex protein (Roc) domain followed by a C terminus of Roc (Cor) domain, which mediates homodimerization (19). In eukaryotes, these domains are commonly followed C terminally by a kinase domain with similarity to the tyrosine kinase-like (TKL) group of kinases (3, 26, 29). In Dictyostelium, other ROCO proteins function in cyclic GMP signaling (8, 35) and cytokinesis (2), and a total of 11 predicted ROCO proteins are present in the genome, 10 of which, including QkgA, encode kinase domains predicted to be catalytically active (17). The human genome encodes two ROCO kinases, which are expressed in a wide range of tissues (25, 40). Little is known regarding the physiological functions of these proteins, although the ROCO protein LRRK2 is implicated in a dominantly inherited form of Parkinson''s disease (40) and negatively regulates neurite growth in rat cortical cultures (28).In this report, we show that, like aprA⁻ and cfaD⁻ cells, qkgA⁻ cells proliferate to a higher cell density than the wild type and tend to be multinucleate. Additionally, we show that qkgA⁻ cells are insensitive to exogenous AprA and CfaD, indicating that QkgA is required for AprA and CfaD signal transduction.     

Secreted Versus Membrane-anchored Collagenases: RELATIVE ROLES IN FIBROBLAST-DEPENDENT COLLAGENOLYSIS AND INVASION*

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13^−/−, Mmp8^−/−, Mmp2^−/−, Mmp9^−/−, Mmp14^−/− and Mmp16^−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14^−/− or Mmp16^−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14^−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.In the postnatal state, fibroblasts are normally embedded in a self-generated three-dimensional connective tissue matrix composed largely of type I collagen, the major extracellular protein found in mammals (1–3). Type I collagen not only acts as a structural scaffolding for the associated mesenchymal cell populations but also regulates gene expression and cell function through its interactions with collagen binding integrins and discoidin receptors (2, 4). Consistent with the central role that type I collagen plays in defining the structure and function of the extracellular matrix, the triple-helical molecule is resistant to almost all forms of proteolytic attack and can display a decades-long half-life in vivo (4–6). Nonetheless, fibroblasts actively remodel type I collagen during wound healing, inflammation, or neoplastic states (2, 7–13).To date type I collagenolytic activity is largely confined to a small subset of fewer than 10 proteases belonging to either the cysteine proteinase or matrix metalloproteinase (MMP)² gene families (4, 14–18). As all collagenases are synthesized as inactive zymogens, complex proteolytic cascades involving serine, cysteine, metallo, and aspartyl proteinases have also been linked to collagen turnover by virtue of their ability to mediate the processing of the pro-collagenases to their active forms (13, 15, 19). After activation, each collagenase can then cleave native collagen within its triple-helical domain, thus precipitating the unwinding or “melting” of the resulting collagen fragments at physiologic temperatures (4, 15). In turn, the denatured products (termed gelatin) are susceptible to further proteolysis by a broader class of “gelatinases” (4, 15). Collagen fragments are then either internalized after binding to specific receptors on the cell surface or degraded to smaller peptides with potent biological activity (20–24).Previous studies by our group as well as others have identified MMPs as the primary effectors of fibroblast-mediated collagenolysis (20, 25, 26). Interestingly, adult mouse fibroblasts express at least six MMPs that can potentially degrade type I collagen, raising the possibility of multiple compensatory networks that are designed to preserve collagenolytic activity (25). Four of these collagenases belong to the family of secreted MMPs, i.e. MMP-13, MMP-8, MMP-2, and MMP-9, whereas the other two enzymes are members of the membrane-type MMP subgroup, i.e. MMP-14 (MT1-MMP) and MMP-16 (MT3-MMP) (13, 27–29). From a functional perspective, the specific roles that can be assigned to secreted versus membrane-anchored collagenases remain undefined. As such, fibroblasts were isolated from either wild-type mice or mice harboring loss-of-function deletions in each of the major secreted and membrane-anchored collagenolytic genes, and the ability of the cells to degrade type I collagen was assessed. Herein, we demonstrate that fibroblasts mobilize either secreted or membrane-anchored MMPs to effectively degrade type I collagen in qualitatively and quantitatively distinct fashions. However, under conditions where fibroblasts use either secreted and membrane-anchored MMPs to exert quantitatively equivalent collagenolytic activity, only MT1-MMP plays a required role in supporting a collagen-invasive phenotype. These data establish a new paradigm wherein secreted collagenases are functionally limited to bulk collagenolytic processes, whereas MT1-MMP uniquely arms the fibroblast with a focalized degradative activity that mediates subjacent collagenolysis as well as invasion.     

Deletion of the Chloride Transporter Slc26a7 Causes Distal Renal Tubular Acidosis and Impairs Gastric Acid Secretion

SLC26A7 (human)/Slc26a7 (mouse) is a recently identified chloride-base exchanger and/or chloride transporter that is expressed on the basolateral membrane of acid-secreting cells in the renal outer medullary collecting duct (OMCD) and in gastric parietal cells. Here, we show that mice with genetic deletion of Slc26a7 expression develop distal renal tubular acidosis, as manifested by metabolic acidosis and alkaline urine pH. In the kidney, basolateral Cl⁻/HCO₃⁻ exchange activity in acid-secreting intercalated cells in the OMCD was significantly decreased in hypertonic medium (a normal milieu for the medulla) but was reduced only mildly in isotonic medium. Changing from a hypertonic to isotonic medium (relative hypotonicity) decreased the membrane abundance of Slc26a7 in kidney cells in vivo and in vitro. In the stomach, stimulated acid secretion was significantly impaired in isolated gastric mucosa and in the intact organ. We propose that SLC26A7 dysfunction should be investigated as a potential cause of unexplained distal renal tubular acidosis or decreased gastric acid secretion in humans.The collecting duct segment of the distal kidney nephron plays a major role in systemic acid base homeostasis by acid secretion and bicarbonate absorption. The acid secretion occurs via H⁺-ATPase and H-K-ATPase into the lumen and bicarbonate is absorbed via basolateral Cl⁻/HCO₃⁻ exchangers (1–4). The tubules, which are located within the outer medullary region of the kidney collecting duct (OMCD),² have the highest rate of acid secretion among the distal tubule segments and are therefore essential to the maintenance of acid base balance (2).The gastric parietal cell is the site of generation of acid and bicarbonate through the action of cytosolic carbonic anhydrase II (5, 6). The intracellular acid is secreted into the lumen via gastric H-K-ATPase, which works in conjunction with a chloride channel and a K⁺ recycling pathway (7–10). The intracellular bicarbonate is transported to the blood via basolateral Cl⁻/HCO₃⁻ exchangers (11–14).SLC26 (human)/Slc26 (mouse) isoforms are members of a conserved family of anion transporters that display tissue-specific patterns of expression in epithelial cells (15–24). Several SLC26 members can function as chloride/bicarbonate exchangers. These include SLC26A3 (DRA), SLC26A4 (pendrin), SLC26A6 (PAT1 or CFEX), SLC26A7, and SLC26A9 (25–31). SLC26A7 and SLC26A9 can also function as chloride channels (32–34).SLC26A7/Slc26a7 is predominantly expressed in the kidney and stomach (28, 29). In the kidney, Slc26a7 co-localizes with AE1, a well-known Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of (acid-secreting) A-intercalated cells in OMCD cells (29, 35, 36) (supplemental Fig. 1). In the stomach, Slc26a7 co-localizes with AE2, a major Cl⁻/HCO₃⁻ exchanger, on the basolateral membrane of acid secreting parietal cells (28). To address the physiological function of Slc26a7 in the intact mouse, we have generated Slc26a7 ko mice. We report here that Slc26a7 ko mice exhibit distal renal tubular acidosis and impaired gastric acidification in the absence of morphological abnormalities in kidney or stomach.     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.