Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Global Analysis of Cdc14 Dephosphorylation Sites Reveals Essential Regulatory Role in Mitosis and Cytokinesis

Degradation of the M phase cyclins triggers the exit from M phase. Cdc14 is the major phosphatase required for the exit from the M phase. One of the functions of Cdc14 is to dephosphorylate and activate the Cdh1/APC/C complex, resulting in the degradation of the M phase cyclins. However, other crucial targets of Cdc14 for mitosis and cytokinesis remain to be elucidated. Here we systematically analyzed the positions of dephosphorylation sites for Cdc14 in the budding yeast Saccharomyces cerevisiae. Quantitative mass spectrometry identified a total of 835 dephosphorylation sites on 455 potential Cdc14 substrates in vivo. We validated two events, and through functional studies we discovered that Cdc14-mediated dephosphorylation of Smc4 and Bud3 is essential for proper mitosis and cytokinesis, respectively. These results provide insight into the Cdc14-mediated pathways for exiting the M phase.All cells proliferate following a fixed, highly coordinated cycle. Mitosis especially requires elaborate coordination for proper chromosome segregation, mitotic spindle disassembly, and cytokinesis. Much of this activity is facilitated by numerous, diverse phosphorylation and dephosphorylation signals that orchestrate the precise progression of M phase.Prior to mitosis, sister chromatids resulting from DNA replication during S phase are held together by the cohesion complex. Then, during prophase, chromosomes are condensed by the condensin (Smc2/4) complex (1) and microtubules are remodeled to form the mitotic spindle (2). Subsequently, in metaphase, the microtubules of the spindle apparatus attach to the chromosome kinetochores (3) and dissolution of the sister chromatids is triggered by the separase-mediated cleavage of cohesin (4, 5). Finally, Cdc14, Cdh1, and APC/C work together in telophase to degrade the M phase cyclins (6), promote decondensation of chromosomes (7), and finish cytokinesis (8, 9).Cdc14, a dual-specificity phosphatase that removes the phosphate group on both phosphotyrosine and phosphoserine/threonine residues (10), is required for mitosis (11, 12). Specifically, Cdc14 function is essential in late M phase: cells carrying a defective mutation arrest in telophase (13), whereas overexpression of Cdc14 results in G1 arrest (12). Cdc14 triggers mitotic cyclin-dependent kinase (CDK)¹ inactivation, enabling cells to exit mitosis through dephosphorylation and activation of the inhibitors of CDKs. At interphase, Cdc14 is a subunit of the mitotic exit network (14–17), which usually localizes to the nucleolus. However, the Cdc14 early anaphase release network initiates the release of Cdc14 from its inhibitor, Net1/Cfi1 (18), and the mitotic exit network promotes further release of Cdc14 from its inhibitor, allowing it to spread into the nucleus and cytoplasm, where it dephosphorylates its major targets (8, 9), leading to exit from mitosis. In addition to this essential role in late M phrase, Cdc14 substrates have also been identified in other stages of the cell cycle (19).Cdc14 putatively regulates 27 proteins (19–22). Some studies have documented the substrates of Cdc14 via in vitro phosphatase assay, whereas others have provided in vivo evidence. However, dephosphorylation sites have been identified for only five of the target proteins (17, 22–25), suggesting that spurious relationships cannot be ruled out. Also, experiments have not been carried out to demonstrate whether these modifications entail direct or indirect regulation. Therefore, our understanding of Cdc14 function and regulation during mitosis in metazoans is incomplete. Conceivably, Cdc14 may regulate many more substrates involved in aspects of chromosome condensation and cytokinesis. To examine this possibility we performed a systematic phosphoproteomic screen to identify new in vivo pathways regulated by Cdc14. Using this approach, we identified both known and potentially novel substrates of Cdc14, as well as their dephosphorylation sites. Many potentially novel substrates are physically associated with Cdc14 in public databases. We also provide biochemical evidence for direct dephosphorylation of the substrates, characterize the specificity of dephosphorylation in two substrates, Smc4 and Bud3, and further study their regulation and critical role in mitosis and cytokinesis.     

New Insights into How the Rho Guanine Nucleotide Dissociation Inhibitor Regulates the Interaction of Cdc42 with Membranes

The subcellular localization of the Rho family GTPases is of fundamental importance to their proper functioning in cells. The Rho guanine nucleotide dissociation inhibitor (RhoGDI) plays a key regulatory role by influencing the cellular localization of Rho GTPases and is essential for the transforming activity of oncogenic forms of Cdc42. However, the mechanism by which RhoGDI helps Cdc42 to undergo the transition between a membrane-associated protein and a soluble (cytosolic) species has been poorly understood. Here, we examine how RhoGDI influences the binding of Cdc42 to lipid bilayers. Despite having similar affinities for the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42 in solution, we show that when RhoGDI interacts with Cdc42 along the membrane surface, it has a much higher affinity for GDP-bound Cdc42 compared with its GTP-bound counterpart. Interestingly, the rate for the dissociation of Cdc42·RhoGDI complexes from membranes is unaffected by the nucleotide-bound state of Cdc42. Moreover, the membrane release of Cdc42·RhoGDI complexes occurs at a similar rate as the release of Cdc42 alone, with the major effect of RhoGDI being to impede the re-association of Cdc42 with membranes. These findings lead us to propose a new model for how RhoGDI influences the ability of Cdc42 to move between membranes and the cytosol, which highlights the role of the membrane in helping RhoGDI to distinguish between the GDP- and GTP-bound forms of Cdc42 and holds important implications for how it functions as a key regulator of the cellular localization and signaling activities of this GTPase.The Rho family GTPases are a tightly regulated class of signaling proteins that controls a number of important cellular processes. Known most prominently for their ability to remodel the actin cytoskeleton in mammalian cells (1–3), members of this GTPase family have been shown to play essential roles in cell migration, epithelial cell polarization, phagocytosis, and cell cycle progression (4–11). The Rho family member Cdc42 was discovered for its essential role in bud formation in Saccharomyces cerevisiae (12). However, after its identification in higher organisms (13), Cdc42 has been implicated in a diverse array of signaling pathways including those involved in the regulation of cell growth and in the induction of malignant transformation (14). Indeed, point mutations which enable Cdc42 to undergo the spontaneous exchange of GDP for GTP cause NIH3T3 cells to form colonies in soft agar and grow in low serum, two hallmarks of cellular transformation (15). The introduction of activated Cdc42 mutants into nude mice gives rise to tumor formation (16). Moreover, cellular transformation by oncogenic Ras, one of the most commonly mutated proteins in human cancers, requires the activation of Cdc42 (17).At the molecular level, there are a number of mechanisms that possibly contribute to the roles played by Cdc42 in cell growth control and cellular transformation. These include the ability of Cdc42 to activate the c-Jun NH₂-terminal kinase and p38/Mpk2 signaling pathways (18–20) as well as spatially regulate proteins implicated in the establishment of microtubule-dependent cell polarity including glycogen synthase kinase-3β and adenomatous polyposis coli (21), extend the lifetime of epidermal growth factor receptor-signaling activities by sequestering Cbl, a ubiquitin E3 ligase (22), and influence intracellular trafficking events (23, 24). To mediate such a wide range of cellular responses, two parameters must be properly regulated; that is, the activation state of Cdc42 and its subcellular localization. As is the case with other GTPases, the activation of Cdc42 occurs as an outcome of GDP-GTP exchange, which then enables it to undergo high affinity interactions with effector proteins (25–27). Upon the hydrolysis of GTP to GDP, Cdc42 is converted back to a signaling-inactive state. Two families of proteins work in opposing fashion to regulate the GTP-binding/GTPase cycle of Cdc42. GTPase-activating proteins recognize the GTP-bound form of Cdc42 and accelerate the hydrolysis of GTP to GDP, rendering Cdc42 inactive (28, 29). Guanine nucleotide exchange factors (GEFs)² stimulate the dissociation of GDP from Cdc42, thereby promoting the formation of its signaling-active, GTP-bound state (29, 30).Of equal importance to its activation status is the spatial regulation of Cdc42. This is highly contingent on the particular cellular membranes that serve as sites of binding and/or recruitment of Cdc42 (31–33). The vast majority of in vitro studies performed on Cdc42 have been carried out in the absence of lipids, which is an important omission considering that virtually all of the physiological functions of Cdc42 occur on a membrane surface (34). Cdc42, along with most other Rho family GTPases, undergoes a series of carboxyl-terminal modifications which result in the covalent attachment of a 20-carbon geranylgeranyl lipid anchor (35–37). Directly preceding this lipid tail is a sequence of basic residues that further stabilizes the association of Cdc42 with the membrane surface (31, 33, 38). A ubiquitously expressed 22-kDa protein called Rho guanine nucleotide dissociation inhibitor (RhoGDI) was found to form a soluble (cytosolic) complex with Cdc42 and other Rho GTPases and to apparently promote their release from membranes (39, 40). RhoGDI was originally discovered and named for its ability to block the GEF- and EDTA-stimulated nucleotide exchange activity of Rho family GTPases (39, 41, 42) and then subsequently shown to inhibit the GTP-hydrolytic activity of Cdc42 (43) and to be capable of interacting with the GDP- and GTP-bound forms of Cdc42 in solution with equal affinity (44). The x-ray crystal structure of a complex between RhoGDI and Cdc42-GDP revealed two types of binding interactions (45). An amino-terminal regulatory arm of RhoGDI was shown to form a helix-loop-helix motif that binds to both of the switch domains of Cdc42, leading to the inhibition of GTP hydrolysis and GDP dissociation (45, 46). The carboxyl-terminal two-thirds of RhoGDI assumes an immunoglobulin-like domain, forming a hydrophobic pocket that in effect provides a membrane substitute for the geranylgeranyl moiety of Cdc42. After release from membranes, the lipid anchor of Cdc42 binds in the hydrophobic pocket of RhoGDI, thereby helping to maintain Cdc42 in solution (45–47).Prior work from our laboratory has demonstrated an essential role for RhoGDI in Cdc42-mediated cellular transformation. Based on the x-ray crystal structure for the Cdc42·RhoGDI complex, Arg-66 of Cdc42 makes multiple contacts with RhoGDI. When this residue was changed to alanine, Cdc42 was unable to bind to RhoGDI but was still capable of interacting with its other regulatory and effector proteins. Interestingly, when the R66A mutant of Cdc42 was examined in the constitutively active Cdc42(F28L) background, the resulting Cdc42 double mutant was no longer able to transform cells (48). Knocking down RhoGDI by small interfering RNA also blocked transformation by Cdc42. These findings highlighted a key role for RhoGDI in the ability of Cdc42 to stimulate signaling pathways of importance to cellular transformation, presumably by influencing the membrane association of Cdc42 and ensuring its proper cellular localization.In the present study we have set out to better understand how RhoGDI regulates the signaling functions of Cdc42 and, in particular, how RhoGDI affects the association of Cdc42 with membranes. We show how the membrane plays a previously unappreciated role in allowing RhoGDI to distinguish between the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42. By assaying the binding of Cdc42 to insect cell membranes and compositionally defined liposomes through different approaches including a sensitive, real-time fluorescence resonance energy transfer (FRET) readout, we have been able to establish how RhoGDI influences the ability of Cdc42 to transition between a membrane-bound and soluble species. This has led us to propose a new mechanism describing how RhoGDI performs its important regulatory function.     

Systems-wide Analysis of K-Ras,Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.The Ras oncoproteins are small monomeric GTPases that transduce mitogenic signals from cell surface receptor tyrosine kinases (RTKs) to intracellular serine/threonine kinases. Approximately thirty percent of human tumors harbor a somatic gain-of-function mutation in one of three RAS genes, resulting in the constitutive activation of Ras signaling and the aberrant hyperactivation of growth-promoting effector pathways (1). Designing therapeutic agents that directly target Ras has been challenging (2, 3), and thus clinical development efforts have focused on targeting effector pathways downstream of Ras. The Raf-MEK-ERK and PI3K-Akt effector pathways have been extensively studied and several small molecule inhibitors targeting these pathways are currently under clinical evaluation (4, 5). However, biochemical studies and mouse models indicate that several additional effector pathways are essential for Ras-driven transformation and tumorigenesis (6–11). Hence, a comprehensive characterization of these effector pathways may reveal additional druggable targets.The Rho GTPase Cdc42 lies downstream of Ras (12–14) and regulates many cellular processes that are commonly perturbed in cancer, including migration, polarization, and proliferation (15) (Fig. 1A). Importantly, Cdc42 is overexpressed in several types of human cancer (16–20) and is required for Ras-driven cellular transformation (13, 21, 22). Recent studies show that genetic ablation of Cdc42 impairs Ras-driven tumorigenesis (13), indicating the potential of Cdc42 and its effectors as drug targets in Ras mutant tumors.Open in a separate windowFig. 1.Experimental workflow. A, K-Ras is a small GTPase that regulates the activity of a variety of downstream proteins including the Rho GTPase Cdc42. The PAK4 serine/threonine kinase is a direct effector of Cdc42 and regulates actin reorganization, microtubule stability, and cell polarity. B, To measure large-scale phosphorylation changes induced by constitutive K-Ras or Cdc42 signaling or PAK4 ablation, the quantitative label-free PTMscan® approach was employed (Cell Signaling Technology). Briefly, for each condition extracted proteins were digested with trypsin and separated from non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Three phosphorylation motif antibodies were used serially to isolate phosphorylated peptides in independent immunoaffinity purifications (CDK substrate motif [K R]-pS-P-X-[K R], CK substrate motif pT-[D E]-X-[D E], PKD substrate motif l-X-R-X-X-p[S T]). The samples were run in duplicate and tandem mass spectra were collected with an LTQ-Orbitrap hybrid mass spectrometer. pLPC is an empty vector control.In particular, the p21-activated kinases (PAKs) are Cdc42 effectors that have generated significant interest (23, 24), as they are central components of key oncogenic signaling pathways and regulate cytoskeletal organization, cell migration, and nuclear signaling (25). The PAK family is comprised of six members and is subdivided into two groups (Groups I and II) based on sequence and structural homology. Group I PAKs (PAK1–3) are relatively well characterized, however, much less is known regarding the function and regulation of Group II PAKs (PAK4–6). The kinase domains of Group I and II PAKs share only about 50% identity, suggesting the two groups may recognize distinct substrates and govern unique cellular processes (26).The Group II PAK family member PAK4 is of particular interest as it is overexpressed or genetically amplified in several lung, colon, prostate, pancreas, and breast tumor cell lines and samples (26–30). Furthermore, functional studies have implicated PAK4 in cell transformation, cell invasion, and migration (27, 31). Xenograft studies in athymic mice show an important role for PAK4 in mediating Cdc42- or K-Ras-driven tumor formation, highlighting a critical role for Pak4 downstream of these GTPases (32). Given its roles in transformation, tumorigenesis, and oncogenic signaling, there is significant interest in targeting PAK4 therapeutically (23). PAK4 binds and phosphorylates several proteins involved in cytoskeletal organization and apoptosis, including Lim domain kinase 1 (LIMK1) (33), guanine nucleotide exchange factor-H1 (GEF-H1) (34), Raf-1 (35), and Bad (36). However, the Group I PAK family member PAK1 also phosphorylates several of these PAK4 targets (37). Thus, there remains a need to identify robust and selective pharmacodynamic biomarkers for PAK4 inhibition.Despite the importance of PAK4 and its upstream regulators in cancer development, few studies have sought to comprehensively characterize the spectrum of K-Ras, Cdc42, or PAK4 mediated phosphorylation signaling (37–39). Recent developments in mass spectrometry allow the in-depth identification and quantitation of thousands of phosphorylation sites (40–43). The majority of large-scale efforts have aimed to identify the basal phosphoproteomes of different species (44, 45) or tissues (46) to characterize global steady-state phosphorylation. However, this methodology can also be applied to quantify perturbed phosphorylation regulation in cancer signaling pathways (40, 47–49), and has the potential to reveal novel biomarkers of oncogenic signaling.In this study, we completed a label-free quantitative analysis of K-Ras, Cdc42, and PAK4 phosphorylation signaling using the PTMScan® method, which has proven as robust and reproducible quantitation technology (50, 51). We quantified phosphorylation levels in wild-type and PAK4 knockout NIH3T3 cells expressing oncogenic K-Ras, activated Cdc42, or an empty vector control to elucidate the molecular pathways and functions modulated by these key signaling proteins. We report relative quantitation of 2152 phosphorylated peptides on 1062 proteins among the different conditions, and find that many of the regulated phosphoproteins are associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. To our knowledge, our study is the first to examine the overlap among signaling networks regulated by K-Ras, Cdc42, and PAK4, and provides a resource for future studies to further interrogate the perturbation of this signaling pathway.     

The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo: A ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE AND Cdc42*

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.Macrophages, terminally differentiated cells of the mononuclear phagocytic lineage, are found throughout the body and play important roles in normal tissue development and immune defense. However, in certain circumstances, excessive recruitment of macrophages has been shown to participate in the progression of several diseases, inflammatory (rheumatoid arthritis) or metabolic (atherosclerosis), as well as in tumor progression (1–3). Importantly expression of colony-stimulating factor-1 (CSF-1),⁴ the most pleiotropic macrophage growth factor, has been correlated with the progression of these disease states (for a review, see Ref. 4). Inhibition of undesirable macrophage recruitment to specific sites in response to CSF-1 is therefore an attractive goal for therapies (5).In addition to stimulating survival, proliferation, and differentiation of monocytes and macrophages, CSF-1 is also a potent chemotactic factor inducing the migration of these cell types (for a review, see Ref. 4). CSF-1 stimulation leads to the rapid production of F-actin-rich protrusions and the spreading and migration of macrophages (4). All CSF-1 effects are mediated through its tyrosine kinase receptor (CSF-1R), which upon activation leads to phosphorylation of tyrosine residues in a number of signaling molecules. Downstream molecules essential for macrophage migration in response to CSF-1 include phosphatidylinositol 3-kinase (PI3K) isoforms β and δ (6, 7). PI3K may potentially regulate migration through the activation of guanine nucleotide exchange factor activity to Rac1 and Cdc42, which are required for CSF-1-elicited protrusions (8, 9) and chemotaxis (10). The major means by which Rac and Cdc42 regulate the Arp2/3 complex is through the Wiskott-Aldrich syndrome protein/Wiskott-Aldrich syndrome verprolin-homologous (WASP/WAVE) family of proteins (11). A Rac1-IRSp53-Abi1-WAVE2 complex has been shown to mediate CSF-1-induced macrophage motility (12, 13), and a unique role for WASP in macrophage chemotaxis to CSF-1, formylmethionylleucylphenylalanine, MCP-1, and MIP-1α has been demonstrated (14, 15). WASP is a hematopoietic cell-specific regulator of Arp2/3-dependent actin remodeling. The catalytically active domain of WASP lies in its C terminus, which is conserved among all WASP/WAVE proteins and contains a VCA (verprolin homology, cofilin-like, and acidic region) domain capable of activating the Arp2/3 complex. The other domains found in WASP can regulate, directly or indirectly, the activity of its VCA domain (for a review, see Ref. 16). Both WASP and N-WASP bind activated Cdc42 through their GTPase-binding domain, which is believed to cause a structural transition that results in dissociation of the intramolecular contacts leaving the VCA domain accessible for Arp2/3 binding (17, 18). In addition, biochemical studies have revealed that several signaling molecules, including WASP-interacting SH3 protein, WASP-interacting protein, Grb2, phosphoinositides, and Src family kinases, activate N-WASP (for reviews, see Refs. 16 and 19). Phosphorylation of WASP has also been proposed to activate Arp2/3-mediated actin polymerization in vitro (20–22).Recently different probes have been developed that detect a conformational change in N-WASP and therefore reflect its activation (23–25). Using either a fluorescence resonance energy transfer (FRET)-based biosensor that detects a conformational change in N-WASP (23, 24) or antibodies that can only bind to the open conformation of N-WASP (25), N-WASP has been shown to be activated in response to epidermal growth factor in HEK293 cells and in MTLn3 carcinoma cells. This activity has been temporally localized to subcellular compartments important for carcinoma cell chemotaxis and invasion (24). We have adapted these approaches to explore the signal transduction pathways responsible for the activation of WASP in vivo.     

Cdc2-mediated Phosphorylation of CLIP-170 Is Essential for Its Inhibition of Centrosome Reduplication

CLIP-170, the founding member of microtubule “plus ends tracking” proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr²⁸⁷ in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.Microtubule dynamics consist of alternating phases of growth and shortening, a pattern of behavior known as dynamic instability (1). This process is tightly regulated by a group of proteins that bind specifically to the plus ends of the growing microtubules (2). Cytoplasmic linker protein (CLIP)³ -170, the founding member of the microtubule plus end family (3), is composed of three separate regions: N terminus, central coiled-coil region, and C terminus. In addition to two conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, the N terminus has three serine-rich regions. The N-terminal domain plays an essential role in microtubule targeting (4), the long central coiled-coil domain is responsible for dimerization of the protein, and the C-terminal region, which contains two zinc-finger domains interferes with microtubule binding by interacting with the N terminus (5). Experiments in a variety of organisms have demonstrated that CLIP-170 plays an important role in microtubule dynamics (6, 7). In addition to its positive role in regulating microtubule growth in both yeast and humans (8, 9), CLIP-170 is involved in recruitment of dynactin to the microtubule plus ends and in linking microtubules to the cortex through Cdc42 and IQGAP (10, 11). The role of CLIP-170 during mitosis was recently examined by loss-of-function approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12, 13).It was previously reported that CLIP-170 is a phosphoprotein and that overall phosphorylation of CLIP-170 affects its microtubule binding ability (14). More recently, metabolic labeling experiments indicated that CLIP-170 is phosphorylated at multiple sites (15). However, individual phosphorylation sites have not been identified. Moreover, the FKBP12-rapamycin-associated protein (FRAP) is the only kinase identified to date for CLIP-170 (15). Therefore, to fully understand the regulation of CLIP-170, it is important to identify individual phosphorylation sites and the responsible kinases. In this communication, we describe a novel kinase/substrate partnership between Cdc2 and CLIP-170. We provide evidence that Cdc2 phosphorylates CLIP-170 at Thr²⁸⁷, and the Cdc2-mediated phosphorylation of CLIP-170 is essential for its localization at microtubule plus ends in the G2 phase and the G2/M transition.     

Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.Protein AMPylation (adenylylation) was recently discovered in bacteria-host interactions where virulence factors catalyze AMPylation using either a conserved Fic domain (e.g., VopS, Vibrio parahaemolyticus (V. para) and IbpA, Histophilus somni) or an adenylyl transferase domain (e.g., DrrA, Legionella pneumophila). These bacterial AMPylation enzymes, or AMPylators, are secreted into the host cells by bacterial secretion systems and transfer AMP from ATP to Tyr or Thr residues of their respective substrates (1–3). In the case of VopS and IbpA, several Rho family GTPases (Rac1, RhoA, and Cdc42) are known substrates and AMPylation disrupts the binding of the GTPase to its downstream effectors, for example, PAK1 (2–6). Considering the conservation of AMPylation domains in both prokaryotic and eukaryotic organisms, we expect that AMPylation plays an important role in a wide range of cellular processes (2, 4, 5, 7–9). Nevertheless, our understanding of this post-translational modification (PTM) is still limited to only a handful of known eukaryotic AMPylation substrates, exclusively belonging to the Rho and Rab GTPase families(10–14). Determining the repertoire of substrates modified by AMPylators will help illuminate both the functional consequences of AMPylation and the mechanistic strategies of pathogens that employ them (6).Significant effort has been devoted to identifying AMPylation substrates. Li et al. systematically investigated the fragmentation patterns of chemically synthesized peptides with Thr, Ser, and Tyr AMPylation using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). They detected AMPylation sites with high confidence and selectively scanned AMPylated peptides in protein mixtures (10). Hao et al. produced a polyclonal antibody that specifically recognized proteins with AMPylation at threonine residues (11). Grammel et al. synthesized an ATP analog, N⁶pATP (N⁶-propargyl adenosine-5′-triphophate), which allows the labeling of AMPylated proteins with azide-functionalized fluorescein or a cleavable biotin enrichment tag (ortho-hydroxy-azidoethoxy-azobiotin) based on copper-catalyzed azide-alkyne cycloaddition (CuAAC)¹. The identification of new substrates for VopS in HeLa cell lysates was explored by a combination of AMP-specific pull-down and LC-MS (12). Using the same approach, Lewallen et al. tried to identify the substrates of VopS in MCF7 cell extracts by employing a commercial N⁶-(6-amino)hexyl-ATP-5-carboxyl-fluorescein (F1-ATP) and anti-fluorescein antibody(13). With these efforts combined, four potential new VopS substrates have been identified (SCCA2, NAGK, NME1, and PFKP), though not yet confirmed. These approaches might miss substrates because of temporal and spatial expression or low abundance in cell lysate, poor recognition by the capture molecules or loss during pull-down procedures (12, 14).Protein microarrays offer a promising approach to identify candidate substrates because they display thousands of unique proteins in a high-throughput and reproducible format (15–17). However, producing arrays with consistent levels of well-folded proteins is challenging because of limitations of protein production, purification, and storage, particularly for mammalian proteins (18).To circumvent these limitations, cell-free protein arrays, which do not require protein purification, have been developed over the past decade (19–22). These methods provide rapid and economical approaches of fabricating protein arrays in terms of cost, shelf life, and storage (23, 24). In cell-free protein arrays, a nucleotide template is printed on the slide and used to produce proteins in vitro with cell-free expression systems from several organisms such as E. coli, wheat germ, and rabbit reticulocyte lysate, etc. (24, 25). These proteins can be engineered to contain fusion tags that enable their capture to the array surface with an appropriate agent. Of these cell-free protein array methods, the Nucleic Acid Programmable Protein Array (NAPPA) is the most advanced, having achieved both high-density and high content containing ∼2300–8000 proteins per slide (20, 26, 27). In NAPPA, a plasmid-based cDNA configured to include an epitope tag is printed on a microscope slide along with the corresponding tag-specific binding reagent, such as an anti-tag antibody, and stored. At the time of experimentation, the cDNA is transcribed/translated into recombinant protein and captured/displayed in situ by the binding reagent. Using a rabbit reticulocyte lysate-based cell-free expression system, NAPPA has been applied toward the identification of novel protein-protein interactions and disease-related antibody biomarkers (20, 26, 28, 29). However, cell-free protein arrays have yet to be employed in the study of PTMs.In this work, we established a novel, nonradioactive unbiased AMPylation screening platform by developing a novel click chemistry-based detection assay for use on high-density cell-free protein microarrays displaying human proteins. Labeling AMP-modified substrates covalently with a fluorophore coupled with the use of human ribosomal machinery and chaperones to produce proteins achieved much higher sensitivity and signal to noise (S/N) ratio compared with previous studies. We screened 10,000 human proteins with two bacterial pathogen AMPylators, VopS and IbpAFic2, identifying more than twenty new substrates each. Two novel Rho GTPases (Rac2 and Rac3) were validated in vivo as substrates of the virulence factor VopS in HEK293T cells during V. para infection. Using mass spectrometry, we verified that a non-GTPase protein, ARHGDIB/LyGDI, was AMPylated by VopS on its threonine 51, which is located in a highly regulated part of this protein. This modification inhibited phosphorylation of LyGDI by Src kinase in vitro. Finally, the identification of these new targets allowed us to build the first bacteria-host interaction AMPylation network and may reveal signaling interactions that could potentially be important for bacterial pathogenesis in the future functional studies.     

Intersectin-2L Regulates Caveola Endocytosis Secondary to Cdc42-mediated Actin Polymerization

Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH_ITSN-2L) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The “catalytically dead” DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH_ITSN-2L-transfected cells compared with control and 1 μm jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.The polymerization of actin has a central role in clathrin- and caveola-mediated endocytosis (1). Studies have shown a number of protein-protein interactions that suggest a functional relationship between the actin cytoskeleton and endocytic machinery; however, the underlying mechanisms remain unclear. ITSN-2L,² a multifunctional domain protein with two Epsin 15 homology domains, a central coiled-coil region followed by five consecutive Src homology 3 domains, a Dbl homology (DH), a pleckstrin homology (PH), and finally a C2 domain, interacts via the Src homology 3 region with the ubiquitously expressed neural Wiskott-Aldrich syndrome protein (N-WASP) that stimulates actin nucleation through Arp 2/3 complex activation (2). ITSN-2L interaction with N-WASP in turn induces activation of N-WASP in a Cdc42-dependent manner (2, 3). In this way, ITSN-2L on the basis of its DH domain acts as a GEF for the small Rho GTPase Cdc42, similar to its neuronal counterpart ITSN-1L (2, 4). The DH domain of ITSN-2L shows high sequence homology with the corresponding region of ITSN-1L (5), and it possesses all the amino acid residues required for its GEF enzymatic activity (6). Both long ITSN isoforms display immediately distal to the DH domain a PH domain, which may thereby modulate the intrinsic catalytic activity of the DH region (6–8). It has been shown that the PH domain enhances up to 100-fold the DH catalytic activity for some Dbl proteins compared with that measured for DH alone in vitro, whereas for other Dbl proteins the presence of the PH domain negatively regulates GEF activity of the DH region (9). This latter function is apparently mediated by interactions with phosphoinositides (7, 9). However, the PH sequence was shown to be dispensable for GEF activity of ITSN-2L in vitro, but it enhanced the ability to activate Cdc42 in vivo (9). Despite high sequence conservation among Rho GTPases, long ITSN isoforms apparently induce selective activation of Cdc42 due to the overall increasing size of the specificity residues of the GTPases (Cdc42 < Rac1 < RhoA) and the inability of ITSN to accommodate in an analogous position the larger size amino acid chains found in Rac1 and RhoA (10).ITSN-2L, like its alternatively spliced short isoform, is widely expressed in human tissue, and it shows subcellular distribution similar to components of the endocytic machinery (5). In COS-7 cells overexpressing ITSN-2 isoforms, clathrin-mediated transferrin uptake was blocked, consistent with their involvement in the regulation of clathrin-mediated endocytosis (5). By contrast, ITSN-2L overexpression in Jurkat cells stimulated T cell antigen receptor (TCR) internalization, whereas truncated ITSN-2L, deleted for the DH domain, caused significant inhibition of TCR internalization (2). The stimulatory effect of ITSN-2L on TCR endocytosis may be secondary to the ability of ITSN-2L to bind through its Src homology 3 domains the proline-rich domain of N-WASP followed by Cdc42-mediated actin polymerization (2). Although more work is needed to clarify these inconsistencies, both of these studies suggest that ITSN-2L may regulate endocytosis and function cooperatively with N-WASP and Cdc42 to link WASP-mediated actin polymerization to the endocytic machinery (2).Live cultured fibroblast imaging showed that actin polymerization as regulated by the WASP-Arp2/3 complex participates in the late stage of clathrin-mediated endocytosis (11). Therefore, we reasoned that ITSN-2L, as a specific activator of Cdc42, may be essential for actin cytoskeleton polymerization and caveola internalization in ECs. ECs are particularly rich in caveola, and caveola-mediated endocytosis is a fundamental step in mediating the transcytosis of proteins (12, 13), but the mechanisms of caveola-mediated endocytosis and the essential proteins involved remain enigmatic. In this study, we addressed the role of ITSN-2L in the mechanism of caveola internalization in ECs. Our data employing morphological, biochemical, and functional approaches show that ITSN-2L on the basis of Cdc42-mediated spatial actin polymerization is required in the mechanism of caveola-mediated endocytosis.     

Aberrant Splice Variants of HAS1 (Hyaluronan Synthase 1) Multimerize with and Modulate Normally Spliced HAS1 Protein: A POTENTIAL MECHANISM PROMOTING HUMAN CANCER*

Most human genes undergo alternative splicing, but aberrant splice forms are hallmarks of many cancers, usually resulting from mutations initiating abnormal exon skipping, intron retention, or the introduction of a new splice sites. We have identified a family of aberrant splice variants of HAS1 (the hyaluronan synthase 1 gene) in some B lineage cancers, characterized by exon skipping and/or partial intron retention events that occur either together or independently in different variants, apparently due to accumulation of inherited and acquired mutations. Cellular, biochemical, and oncogenic properties of full-length HAS1 (HAS1-FL) and HAS1 splice variants Va, Vb, and Vc (HAS1-Vs) are compared and characterized. When co-expressed, the properties of HAS1-Vs are dominant over those of HAS1-FL. HAS1-FL appears to be diffusely expressed in the cell, but HAS1-Vs are concentrated in the cytoplasm and/or Golgi apparatus. HAS1-Vs synthesize detectable de novo HA intracellularly. Each of the HAS1-Vs is able to relocalize HAS1-FL protein from diffuse cytoskeleton-anchored locations to deeper cytoplasmic spaces. This HAS1-Vs-mediated relocalization occurs through strong molecular interactions, which also serve to protect HAS1-FL from its otherwise high turnover kinetics. In co-transfected cells, HAS1-FL and HAS1-Vs interact with themselves and with each other to form heteromeric multiprotein assemblies. HAS1-Vc was found to be transforming in vitro and tumorigenic in vivo when introduced as a single oncogene to untransformed cells. The altered distribution and half-life of HAS1-FL, coupled with the characteristics of the HAS1-Vs suggest possible mechanisms whereby the aberrant splicing observed in human cancer may contribute to oncogenesis and disease progression.About 70–80% of human genes undergo alternative splicing, contributing to proteomic diversity and regulatory complexities in normal development (1). About 10% of mutations listed so far in the Human Gene Mutation Database (HGMD) of “gene lesions responsible for human inherited disease” were found to be located within splice sites. Furthermore, it is becoming increasingly apparent that aberrant splice variants, generated mostly due to splicing defects, play a key role in cancer. Germ line or acquired genomic changes (mutations) in/around splicing elements (2–4) promote aberrant splicing and aberrant protein isoforms.Hyaluronan (HA)³ is synthesized by three different plasma membrane-bound hyaluronan synthases (1, 2, and 3). HAS1 undergoes alternative and aberrant intronic splicing in multiple myeloma, producing truncated variants termed Va, Vb, and Vc (5, 6), which predicted for poor survival in a cohort of multiple myeloma patients (5). Our work suggests that this aberrant splicing arises due to inherited predispositions and acquired mutations in the HAS1 gene (7). Cancer-related, defective mRNA splicing caused by polymorphisms and/or mutations in splicing elements often results in inactivation of tumor suppressor activity (e.g. HRPT2 (8, 9), PTEN (10), MLHI (11–14), and ATR (15)) or generation of dominant negative inhibitors (e.g. CHEK2 (16) and VWOX (17)). In breast cancer, aberrantly spliced forms of progesterone and estrogen receptors are found (reviewed in Ref. 3). Intronic mutations inactivate p53 through aberrant splicing and intron retention (18). Somatic mutations with the potential to alter splicing are frequent in some cancers (19–25). Single nucleotide polymorphisms in the cyclin D1 proto-oncogene predispose to aberrant splicing and the cyclin D1b intronic splice variant (26–29). Cyclin D1b confers anchorage independence, is tumorogenic in vivo, and is detectable in human tumors (30), but as yet no clinical studies have confirmed an impact on outcome. On the other hand, aberrant splicing of HAS1 shows an association between aberrant splice variants and malignancy, suggesting that such variants may be potential therapeutic targets and diagnostic indicators (19, 31–33). Increased HA expression has been associated with malignant progression of multiple tumor types, including breast, prostate, colon, glioma, mesothelioma, and multiple myeloma (34). The three mammalian HA synthase (HAS) isoenzymes synthesize HA and are integral transmembrane proteins with a probable porelike structural assembly (35–39). Although in humans, the three HAS genes are located on different chromosomes (hCh19, hCh8, and hCh16, respectively) (40), they share a high degree of sequence homology (41, 42). HAS isoenzymes synthesize a different size range of HA molecules, which exhibit different functions (43, 44). HASs contribute to a variety of cancers (45–55). Overexpression of HASs promotes growth and/or metastatic development in fibrosarcoma, prostate, and mammary carcinoma, and the removal of the HA matrix from a migratory cell membrane inhibits cell movement (45, 53). HAS2 confers anchorage independence (56). Our work has shown aberrant HAS1 splicing in multiple myeloma (5) and Waldenstrom''s macroglobulinemia (6). HAS1 is overexpressed in colon (57), ovarian (58), endometrial (59), mesothelioma (60), and bladder cancers (61). A HAS1 splice variant is detected in bladder cancer (61).Here, we characterize molecular and biochemical characteristics of HAS1 variants (HAS1-Vs) (5), generated by aberrant splicing. Using transient transfectants and tagged HAS1 family constructs, we show that HAS1-Vs differ in cellular localization, de novo HA localization, and turnover kinetics, as compared with HAS1-FL, and dominantly influence HAS1-FL when co-expressed. HAS1-Vs proteins form intra- and intermolecular associations among themselves and with HAS1-FL, including covalent interactions and multimer formation. HAS1-Vc supports vigorous cellular transformation of NIH3T3 cells in vitro, and HAS1-Vc-transformed NIH3T3 cells are tumorogenic in vivo.