Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis

Early onset generalized dystonia (DYT1) is an autosomal dominant neurological disorder caused by deletion of a single glutamate residue (torsinA ΔE) in the C-terminal region of the AAA⁺ (ATPases associated with a variety of cellular activities) protein torsinA. The pathogenic mechanism by which torsinA ΔE mutation leads to dystonia remains unknown. Here we report the identification and characterization of a 628-amino acid novel protein, printor, that interacts with torsinA. Printor co-distributes with torsinA in multiple brain regions and co-localizes with torsinA in the endoplasmic reticulum. Interestingly, printor selectively binds to the ATP-free form but not to the ATP-bound form of torsinA, supporting a role for printor as a cofactor rather than a substrate of torsinA. The interaction of printor with torsinA is completely abolished by the dystonia-associated torsinA ΔE mutation. Our findings suggest that printor is a new component of the DYT1 pathogenic pathway and provide a potential molecular target for therapeutic intervention in dystonia.Early onset generalized torsion dystonia (DYT1) is the most common and severe form of hereditary dystonia, a movement disorder characterized by involuntary movements and sustained muscle spasms (1). This autosomal dominant disease has childhood onset and its dystonic symptoms are thought to result from neuronal dysfunction rather than neurodegeneration (2, 3). Most DYT1 cases are caused by deletion of a single glutamate residue at positions 302 or 303 (torsinA ΔE) of the 332-amino acid protein torsinA (4). In addition, a different torsinA mutation that deletes amino acids Phe³²³–Tyr³²⁸ (torsinA Δ323–328) was identified in a single family with dystonia (5), although the pathogenic significance of this torsinA mutation is unclear because these patients contain a concomitant mutation in another dystonia-related protein, ϵ-sarcoglycan (6). Recently, genetic association studies have implicated polymorphisms in the torsinA gene as a genetic risk factor in the development of adult-onset idiopathic dystonia (7, 8).TorsinA contains an N-terminal endoplasmic reticulum (ER)³ signal sequence and a 20-amino acid hydrophobic region followed by a conserved AAA⁺ (ATPases associated with a variety of cellular activities) domain (9, 10). Because members of the AAA⁺ family are known to facilitate conformational changes in target proteins (11, 12), it has been proposed that torsinA may function as a molecular chaperone (13, 14). TorsinA is widely expressed in brain and multiple other tissues (15) and is primarily associated with the ER and nuclear envelope (NE) compartments in cells (16–20). TorsinA is believed to mainly reside in the lumen of the ER and NE (17–19) and has been shown to bind lamina-associated polypeptide 1 (LAP1) (21), lumenal domain-like LAP1 (LULL1) (21), and nesprins (22). In addition, recent evidence indicates that a significant pool of torsinA exhibits a topology in which the AAA⁺ domain faces the cytoplasm (20). In support of this topology, torsinA is found in the cytoplasm, neuronal processes, and synaptic terminals (2, 3, 15, 23–26) and has been shown to bind cytosolic proteins snapin (27) and kinesin light chain 1 (20). TorsinA has been proposed to play a role in several cellular processes, including dopaminergic neurotransmission (28–31), NE organization and dynamics (17, 22, 32), and protein trafficking (27, 33). However, the precise biological function of torsinA and its regulation remain unknown.To gain insights into torsinA function, we performed yeast two-hybrid screens to search for torsinA-interacting proteins in the brain. We report here the isolation and characterization of a novel protein named printor (protein interactor of torsinA) that interacts selectively with wild-type (WT) torsinA but not the dystonia-associated torsinA ΔE mutant. Our data suggest that printor may serve as a cofactor of torsinA and provide a new molecular target for understanding and treating dystonia.     

Sindbis Virus Induces Apoptosis through a Caspase-Dependent,CrmA-Sensitive Pathway

Sindbis virus infection of cultured cells and of neurons in mouse brains leads to programmed cell death exhibiting the classical characteristics of apoptosis. Although the mechanism by which Sindbis virus activates the cell suicide program is not known, we demonstrate here that Sindbis virus activates caspases, a family of death-inducing proteases, resulting in cleavage of several cellular substrates. To study the role of caspases in virus-induced apoptosis, we determined the effects of specific caspase inhibitors on Sindbis virus-induced cell death. CrmA (a serpin from cowpox virus) and zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) inhibited Sindbis virus-induced cell death, suggesting that cellular caspases facilitate apoptosis induced by Sindbis virus. Furthermore, CrmA significantly increased the rate of survival of infected mice. These inhibitors appear to protect cells by inhibiting the cellular death pathway rather than impairing virus replication or by inhibiting the nsP2 and capsid viral proteases. The specificity of CrmA indicates that the Sindbis virus-induced death pathway is similar to that induced by Fas or tumor necrosis factor alpha rather than being like the death pathway induced by DNA damage. Taken together, these data suggest a central role for caspases in Sindbis virus-induced apoptosis.Sindbis virus is an alphavirus of the Togaviridae family which causes encephalitis in mice and thus serves as a model for closely related human encephalitic viruses. Infection of a variety of cultured cell types with Sindbis virus triggers programmed cell death (33). The induction of apoptosis in neurons of mouse brains and spinal cords correlates with the neurovirulence of the virus strain and with mortality in mice, suggesting that induction of apoptosis may be a primary cause of death of young mice (34). In support of this hypothesis, overexpressed inhibitors of apoptosis, such as Bcl-2 and IAP, can protect cultured cells from Sindbis virus-induced apoptosis, and Bcl-2 efficiently reduces mortality in mice (17, 31, 32). These findings also raise the possibility that endogenous inhibitors of apoptosis inhibit Sindbis virus-induced cell death, leading to a persistent virus infection (33, 61). Encephalitis and/or a fatal stress response may be a consequence of neuronal apoptosis (21, 59). Alternatively, there may be multiple pathways that work independently to cause fatal disease.A crucial role for the caspase family of cysteine proteases in the execution phase of programmed cell death is supported by genetic (24, 52, 66), biochemical (29, 57), and physiological (25) evidence. A current model proposes a cascade of events by which caspases proteolytically activate other caspases (35, 39, 46). More recent evidence suggests that different death stimuli trigger the activation of a subset of upstream caspases that possess long prodomains at their N termini (3, 41, 62). These prodomains serve to target proteases to specific protein complexes, where the prodomains are removed by proteolysis to produce active proteases. These caspases proteolytically activate other downstream caspases (with shorter prodomains) that cleave key substrates to ultimately produce the characteristic apoptotic phenotype of cell shrinkage, membrane blebbing, chromatin condensation, oligonucleosomal DNA fragmentation, and cell death (42, 53). A growing list of proteolytic substrates of the caspases have been identified, including protein kinase C delta (18), the retinoblastoma tumor suppressor (56), fodrin (12, 38), lamins (30, 47), the nuclear immunophilin FKBP46 (1), Bcl-2 (7), and several autoantigens (5), and they all are cleaved after an aspartate residue (P1 position). The precise role of these cleavage events is not known, but they may either inactivate key cellular functions or produce cleavage products with pro-death activity. The cleavage product of Bcl-2 is potently proapoptotic (7), and cleavage of a novel protein designated DFF was recently shown to trigger DNA fragmentation during apoptosis (36). These proteolytic events also serve as biochemical markers of apoptosis. Furthermore, cell death can be inhibited with pseudosubstrate inhibitors of the caspases, such as the cowpox virus serpin CrmA (19, 48), and synthetic peptides such as zVAD-FMK (67). The key feature of these inhibitors is an aspartate at the P1 position, consistent with their specificity for caspases.A role for caspases in viral infections is suggested by the finding that baculovirus infection activates an apoptotic cysteine protease in insect cells that is inhibited by the virus-encoded caspase inhibitor p35 (2). Similar work with mutant adenoviruses has suggested that the adenovirus protein E1A activates caspase 3 (CPP32), generating cleaved products of poly(ADP-ribose) polymerase (PARP) (4). In addition, PARP cleavage is detected during infection of mouse neuroblastoma cells with Sindbis virus (60). To further study the role of these proteases in Sindbis virus-induced programmed cell death, we confirmed that Sindbis virus activates cellular caspases and demonstrated the participation of a subset of caspases in the execution of the apoptotic process.     

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.     

Evidence of a Role for Phosphatidylinositol 3-Kinase Activation in the Blocking of Apoptosis by Polyomavirus Middle T Antigen

A polyomavirus mutant (315YF) blocked in binding phosphatidylinositol 3-kinase (PI 3-kinase) has previously been shown to be partially deficient in transformation and to induce fewer tumors and with a significant delay compared to wild-type virus. The role of polyomavirus middle T antigen-activated PI 3-kinase in apoptosis was investigated as a possible cause of this behavior. When grown in medium containing 1d-3-deoxy-3-fluoro-myo-inositol to block formation of 3′-phosphorylated phosphatidylinositols, F111 rat fibroblasts transformed by wild-type polyomavirus (PyF), but not normal F111 cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI 3-kinase, stimulated apoptosis in PyF cells but not in normal cells. Activation of Akt, a serine/threonine kinase whose activity has been correlated with regulation of apoptosis, was roughly twofold higher in F111 cells transformed by either wild-type virus or mutant 250YS blocked in binding Shc compared to cells transformed by mutant 315YF. In the same cells, levels of apoptosis were inversely correlated with Akt activity. Apoptosis induced by serum withdrawal in Rat-1 cells expressing a temperature-sensitive p53 was shown to be at least partially p53 independent. Expression of either wild-type or 250YS middle T antigen inhibited apoptosis in serum-starved Rat-1 cells at both permissive and restrictive temperatures for p53. Mutant 315YF middle T antigen was partially defective for inhibition of apoptosis in these cells. The results indicate that unlike other DNA tumor viruses which block apoptosis by inactivation of p53, polyomavirus achieves protection from apoptotic death through a middle T antigen–PI 3-kinase–Akt pathway that is at least partially p53 independent.Programmed cell death occurs during normal development and under certain pathological conditions. In mammalian cells, apoptosis can be induced by a variety of stimuli, including DNA damage (45), virus infection (54, 57), oncogene activation (25), and serum withdrawal (34, 37). Apoptosis can also be blocked by a number of factors, including adenovirus E1B 55- or 19-kDa proteins (9, 16), baculovirus p35 and iap genes (10), Bcl-2 (36, 61), and survival factors (12, 21). DNA tumor viruses have evolved mechanisms that both trigger and inhibit apoptosis. These frequently involve binding and inactivation of tumor suppressor proteins. E7 in some papillomaviruses (22), E1A in adenovirus (31, 43, 64), and large T antigen in simian virus 40 (SV40) (17) bind Rb and/or p300 and lead to upregulation of p53, which is thought to trigger apoptosis in virus-infected cells. The same viruses also inhibit apoptosis by inactivating p53 by various mechanisms (44, 63, 67). In contrast, the mechanism by which polyomavirus interacts with apoptotic pathways in the cell is not known; no direct interaction with p53 by any of the proteins encoded by this virus has been demonstrated (19, 62).The principal oncoprotein of polyomavirus is the middle T antigen. Neoplastic transformation by polyomavirus middle T antigen has as a central feature its association with and activation of members of the Src family of tyrosine kinases p60^c-src (13) and p62^c-yes (42). The major known consequence of these interactions is phosphorylation of middle T antigen on specific tyrosine residues creating binding sites for other signaling proteins. Phosphorylation at tyrosines 250, 315, and 322 promotes binding to Shc (18), the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (59), and phospholipase Cγ-1 (58), respectively. Recognition of multiple signaling pathways emanating from middle T antigen has led to a keen interest in identifying their downstream biochemical effects, which collectively lead to the emergence of neoplastic transformation and presumably underlie the dramatic ability of the virus to induce many kinds of tumors in the mouse.Previous work has shown that the binding of PI 3-kinase to middle T antigen is essential for full transformation of rat fibroblasts in culture (8) and for rapid development of a broad spectrum of tumors in mice (30), for translocation of the GLUT1 transporter (68), and activation of p70 S6 kinase (14). While the mutant 315YF (blocked in PI 3-kinase activation) was able to induce some tumors, it did so at reduced frequencies and with an average latency three times longer than that of either the wild-type virus or a mutant, 250YS, blocked in binding Shc (4, 30). Recent studies have indicated a role of PI 3-kinase in blocking apoptosis in nonviral systems. Growth factor receptors acting through protein tyrosine kinases may prevent apoptosis by activating PI 3-kinase in PC12 cells, T lymphocytes, hematopoietic progenitors, and rat fibroblasts (7, 48, 56, 65, 66). The failure of mutant 315YF to induce full transformation of cells in culture and to induce the rapid development of tumors in mice could therefore be related, at least in part, to a failure to block apoptosis. In this study, we focus on the question of whether middle T antigen–PI 3-kinase interaction is involved in blocking apoptosis in cells transformed by polyomavirus.     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.