Mcl-1 Integrates the Opposing Actions of Signaling Pathways That Mediate Survival and Apoptosis

Mcl-1 is a member of the Bcl2-related protein family that is a critical mediator of cell survival. Exposure of cells to stress causes inhibition of Mcl-1 mRNA translation and rapid destruction of Mcl-1 protein by proteasomal degradation mediated by a phosphodegron created by glycogen synthase kinase 3 (GSK3) phosphorylation of Mcl-1. Here we demonstrate that prior phosphorylation of Mcl-1 by the c-Jun N-terminal protein kinase (JNK) is essential for Mcl-1 phosphorylation by GSK3. Stress-induced Mcl-1 degradation therefore requires the coordinated activity of JNK and GSK3. Together, these data establish that Mcl-1 functions as a site of signal integration between the proapoptotic activity of JNK and the prosurvival activity of the AKT pathway that inhibits GSK3.Mcl-1 is an antiapoptotic member of the Bcl2 family. Gene knockout studies of mice demonstrate that Mcl-1 is essential for embryonic development and for the survival of hematopoietic cells (28-30). Studies of the stress response have demonstrated that Mcl-1 plays an important role in the sensitization of cells to apoptotic signals (1, 11, 25). Thus, exposure to UV radiation causes the rapid degradation of Mcl-1 and the release of proapoptotic partner proteins from Mcl-1 complexes (e.g., Bim). The mechanism of rapid Mcl-1 destruction is mediated by the combined actions of two different pathways. First, the exposure to stress causes phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2α) on the inhibitory site Ser-51 that prevents translation of Mcl-1 mRNA (1, 11, 25). Second, Mcl-1 is rapidly degraded by the ubiquitin-dependent proteasome pathway (27). Together, these pathways cause a rapid reduction in Mcl-1 expression. This loss of Mcl-1 may be a required initial response for the apoptosis of cells exposed to stress (25).The E3 ubiquitin protein ligase Mule/ARF-BP1 contains a BH3 domain that interacts with Mcl-1 and can initiate ubiquitin-dependent degradation of Mcl-1 (39). Recent studies have demonstrated that rapid stress-induced degradation of Mcl-1 is mediated by an alternative pathway involving the E3 ubiquitin protein ligase β-TrCP, which binds a stress-induced phosphodegron created by the phosphorylation of Mcl-1 by glycogen synthase kinase 3 (GSK3) (7, 21). How the exposure to stress causes GSK3-mediated phosphorylation of Mcl-1 is unclear, but GSK3 has been shown to directly phosphorylate Mcl-1 (7, 21). Mcl-1 phosphorylation and degradation may therefore be controlled by the prosurvival AKT pathway, which can negatively regulate GSK3 (7, 21).Mcl-1 is critically involved in the regulation of cell survival and is therefore subject to regulation by multiple mechanisms (26). Thus, Mcl-1 gene expression is regulated by many growth factors and cytokines (26), and Mcl-1 mRNA is regulated by microRNA pathways (24). The Mcl-1 protein is stabilized by binding TCTP (20) and the BH3-only protein Bim (4). In contrast, the BH3-only protein Noxa binds and destabilizes Mcl-1 (4, 36). Moreover, it is established that Mcl-1 is phosphorylated by several protein kinases on sites that may regulate Mcl-1 function. Phosphorylation of human Mcl-1 (hMcl-1) on Ser-64 (a site that is not conserved in other species) may enhance antiapoptotic activity by increasing the interaction of Mcl-1 with Bim, Noxa, and Bak (18). Phosphorylation on Ser-121 and Thr-163 may inhibit the antiapoptotic activity of hMcl-1 (15), and phosphorylation on Thr-163 may increase hMcl-1 protein stability (9). The conserved GSK3 phosphorylation site Ser-159 (and possibly Ser-155) can initiate rapid proteasomal degradation of hMcl-1 (7, 21). Together, these findings suggest that the function of Mcl-1 is very tightly regulated.The results of previous studies have implicated the c-Jun N-terminal protein kinase (JNK) in the regulation of Mcl-1 (15, 18). The purpose of this study was to test whether Mcl-1 is a target of signal transduction by JNK. We demonstrate that a key function of JNK is to prime Mcl-1 for phosphorylation by GSK3. JNK is required for GSK3-mediated degradation of Mcl-1 in response to stress. Coordinated regulation of the stress-activated JNK pathway and the AKT-inhibited GSK3 pathway is therefore required for stress-induced Mcl-1 degradation.     

mTORC1-Activated S6K1 Phosphorylates Rictor on Threonine 1135 and Regulates mTORC2 Signaling

The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that forms two functionally distinct complexes important for nutrient and growth factor signaling. While mTOR complex 1 (mTORC1) regulates mRNA translation and ribosome biogenesis, mTORC2 plays an important role in the phosphorylation and subsequent activation of Akt. Interestingly, mTORC1 negatively regulates Akt activation, but whether mTORC1 signaling directly targets mTORC2 remains unknown. Here we show that growth factors promote the phosphorylation of Rictor (rapamycin-insensitive companion of mTOR), an essential subunit of mTORC2. We found that Rictor phosphorylation requires mTORC1 activity and, more specifically, the p70 ribosomal S6 kinase 1 (S6K1). We identified several phosphorylation sites in Rictor and found that Thr1135 is directly phosphorylated by S6K1 in vitro and in vivo, in a rapamycin-sensitive manner. Phosphorylation of Rictor on Thr1135 did not affect mTORC2 assembly, kinase activity, or cellular localization. However, cells expressing a Rictor T1135A mutant were found to have increased mTORC2-dependent phosphorylation of Akt. In addition, phosphorylation of the Akt substrates FoxO1/3a and glycogen synthase kinase 3α/β (GSK3α/β) was found to be increased in these cells, indicating that S6K1-mediated phosphorylation of Rictor inhibits mTORC2 and Akt signaling. Together, our results uncover a new regulatory link between the two mTOR complexes, whereby Rictor integrates mTORC1-dependent signaling.The mammalian target of rapamycin (mTOR) is an evolutionarily conserved phosphatidylinositol 3-kinase (PI3K)-related Ser/Thr kinase that integrates signals from nutrients, energy sufficiency, and growth factors to regulate cell growth as well as organ and body size in a variety of organisms (reviewed in references 4, 38, 49, and 77). mTOR was discovered as the molecular target of rapamycin, an antifungal agent used clinically as an immunosuppressant and more recently as an anticancer drug (5, 20). Recent evidence indicates that deregulation of the mTOR pathway occurs in a majority of human cancers (12, 18, 25, 46), suggesting that rapamycin analogs may be potent antineoplastic therapeutic agents.mTOR forms two distinct multiprotein complexes, the rapamycin-sensitive and -insensitive mTOR complexes 1 and 2 (mTORC1 and mTORC2), respectively (6, 47). In cells, rapamycin interacts with FKBP12 and targets the FKBP12-rapamycin binding (FRB) domain of mTORC1, thereby inhibiting some of its function (13, 40, 66). mTORC1 is comprised of the mTOR catalytic subunit and four associated proteins, Raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec13 protein 8), PRAS40 (proline-rich Akt substrate of 40 kDa), and Deptor (28, 43, 44, 47, 59, 73, 74). The small GTPase Rheb (Ras homolog enriched in brain) is a key upstream activator of mTORC1 that is negatively regulated by the tuberous sclerosis complex 1 (TSC1)/TSC2 GTPase-activating protein complex (reviewed in reference 35). mTORC1 is activated by PI3K and Ras signaling through direct phosphorylation and inactivation of TSC2 by Akt, extracellular signal-regulated kinase (ERK), and p90 ribosomal protein S6 kinase (RSK) (11, 37, 48, 53, 63). mTORC1 activity is also regulated at the level of Raptor. Whereas low cellular energy levels negatively regulate mTORC1 activity through phosphorylation of Raptor by AMP-activated protein kinase (AMPK) (27), growth signaling pathways activating the Ras/ERK pathway positively regulate mTORC1 activity through direct phosphorylation of Raptor by RSK (10). More recent evidence has also shown that mTOR itself positively regulates mTORC1 activity by directly phosphorylating Raptor at proline-directed sites (20a, 75). Countertransport of amino acids (55) and amino acid signaling through the Rag GTPases were also shown to regulate mTORC1 activity (45, 65). When activated, mTORC1 phosphorylates two main regulators of mRNA translation and ribosome biogenesis, the AGC (protein kinase A, G, and C) family kinase p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and thus stimulates protein synthesis and cellular growth (50, 60).The second mTOR complex, mTORC2, is comprised of mTOR, Rictor (rapamycin-insensitive companion of mTOR), mSin1 (mammalian stress-activated mitogen-activated protein kinase-interacting protein 1), mLST8, PRR5 (proline-rich region 5), and Deptor (21, 39, 58, 59, 66, 76, 79). Rapamycin does not directly target and inhibit mTORC2, but long-term treatment with this drug was shown to correlate with mTORC2 disassembly and cytoplasmic accumulation of Rictor (21, 39, 62, 79). Whereas mTORC1 regulates hydrophobic motif phosphorylation of S6K1, mTORC2 has been shown to phosphorylate other members of the AGC family of kinases. Biochemical and genetic evidence has demonstrated that mTORC2 phosphorylates Akt at Ser473 (26, 39, 68, 70), thereby contributing to growth factor-mediated Akt activation (6, 7, 52). Deletion or knockdown of the mTORC2 components mTOR, Rictor, mSin1, and mLST8 has a dramatic effect on mTORC2 assembly and Akt phosphorylation at Ser473 (26, 39, 79). mTORC2 was also shown to regulate protein kinase Cα (PKCα) (26, 66) and, more recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1) (4, 22). Recent evidence implicates mTORC2 in the regulation of Akt and PKCα phosphorylation at their turn motifs (19, 36), but whether mTOR directly phosphorylates these sites remains a subject of debate (4).Activation of mTORC1 has been shown to negatively regulate Akt phosphorylation in response to insulin or insulin-like growth factor 1 (IGF1) (reviewed in references 30 and 51). This negative regulation is particularly evident in cell culture models with defects in the TSC1/TSC2 complex, where mTORC1 and S6K1 are constitutively activated. Phosphorylation of insulin receptor substrate-1 (IRS-1) by mTORC1 (72) and its downstream target S6K1 has been shown to decrease its stability and lead to an inability of insulin or IGF1 to activate PI3K and Akt (29, 69). Although the mechanism is unknown, platelet-derived growth factor receptor β (PDGF-Rβ) has been found to be downregulated in TSC1- and TSC2-deficient murine embryonic fibroblasts (MEFs), contributing to a reduction of PI3K signaling (80). Interestingly, inhibition of Akt phosphorylation by mTORC1 has also been observed in the presence of growth factors other than IGF-1, insulin, or PDGF, suggesting that there are other mechanisms by which mTORC1 activation restricts Akt activity in cells (reviewed in references 6 and 31). Recent evidence demonstrates that rapamycin treatment causes a significant increase in Rictor electrophoretic mobility (2, 62), suggesting that phosphorylation of the mTORC2 subunit Rictor may be regulated by mTORC1 or downstream protein kinases.Herein, we demonstrate that Rictor is phosphorylated by S6K1 in response to mTORC1 activation. We demonstrate that Thr1135 is directly phosphorylated by S6K1 and found that a Rictor mutant lacking this phosphorylation site increases Akt phosphorylation induced by growth factor stimulation. Cells expressing the Rictor T1135A mutant were found to have increased Akt signaling to its substrates compared to Rictor wild-type- and T1135D mutant-expressing cells. Together, our results suggest that Rictor integrates mTORC1 signaling via its phosphorylation by S6K1, resulting in the inhibition of mTORC2 and Akt signaling.     

Protein Tyrosine Kinase 6 Directly Phosphorylates AKT and Promotes AKT Activation in Response to Epidermal Growth Factor

Protein tyrosine kinase 6 (PTK6) is a nonmyristoylated Src-related intracellular tyrosine kinase. Although not expressed in the normal mammary gland, PTK6 is expressed in a majority of human breast tumors examined, and it has been linked to ErbB receptor signaling and AKT activation. Here we demonstrate that AKT is a direct substrate of PTK6 and that AKT tyrosine residues 315 and 326 are phosphorylated by PTK6. Association of PTK6 with AKT occurs through the SH3 domain of PTK6 and is enhanced through SH2 domain-mediated interactions following tyrosine phosphorylation of AKT. Using Src, Yes, and Fyn null mouse embryonic fibroblasts (SYF cells), we show that PTK6 phosphorylates AKT in a Src family kinase-independent manner. Introduction of PTK6 into SYF cells sensitized these cells to physiological levels of epidermal growth factor (EGF) and increased AKT activation. Stable introduction of active PTK6 into SYF cells also resulted in increased proliferation. Knockdown of PTK6 in the BPH-1 human prostate epithelial cell line led to decreased AKT activation in response to EGF. Our data indicate that in addition to promoting growth factor receptor-mediated activation of AKT, PTK6 can directly activate AKT to promote oncogenic signaling.Protein tyrosine kinase 6 (PTK6; also known as the breast tumor kinase BRK) is an intracellular Src-related tyrosine kinase (9, 48). Human PTK6 was identified in cultured human melanocytes (32) and breast tumor cells (39), while its mouse orthologue was cloned from normal small intestinal epithelial cell RNA (50). Although PTK6 shares overall structural similarity with Src family tyrosine kinases, it lacks an N-terminal myristoylation consensus sequence for membrane targeting (39, 51). As a consequence, PTK6 is localized to different cellular compartments, including the nucleus (14, 15). PTK6 is expressed in normal differentiated epithelial cells of the gastrointestinal tract (34, 42, 51), prostate (14), and skin (51-53). Expression of PTK6 is upregulated in different types of cancers, including breast carcinomas (6, 39, 54), colon cancer (34), ovarian cancer (47), head and neck cancers (33), and metastatic melanoma cells (16). The significance of apparent opposing signaling roles for PTK6 in normal differentiation and cancer is still poorly understood.In human breast tumor cells, PTK6 enhances signaling from members of the ErbB receptor family (10, 29, 30, 36, 40, 49, 54). In the HB4a immortalized human mammary gland luminal epithelial cell line, PTK6 promoted epidermal growth factor (EGF)-induced ErbB3 tyrosine phosphorylation and AKT activation (29). In response to EGF stimulation, PTK6 promoted phosphorylation of the focal adhesion protein paxillin and Rac1-mediated cell migration (10). PTK6 can be activated by the ErbB3 ligand heregulin and promotes activation of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein kinase (MAPK) in breast cancer cells (40). PTK6 can also phosphorylate p190RhoGAP-A and stimulate its activity, leading to RhoA inactivation and Ras activation and thereby promoting EGF-dependent breast cancer cell migration and proliferation (49). Expression of PTK6 has been correlated with ErbB2 expression in human breast cancers (4, 5, 54).AKT (also called protein kinase B) is a serine-threonine kinase that is activated downstream of growth factor receptors (38). It is a key player in signaling pathways that regulate energy metabolism, proliferation, and cell survival (7, 45). Aberrant activation of AKT through diverse mechanisms has been discovered in different cancers (2). AKT activation requires phosphorylation of AKT on threonine residue 308 and serine residue 473. The significance of phosphorylation of AKT on tyrosine residues is less well understood. Src has been shown to phosphorylate AKT on conserved tyrosine residues 315 and 326 near the activation loop (11). Substitution of these two tyrosine residues with phenylalanine abolished AKT kinase activity stimulated by EGF (11). Use of the Src family inhibitor PP2 impaired AKT activation following IGF-1 stimulation of oligodendrocytes (13). The RET/PTC receptor tyrosine kinase that responds to glial cell-line-derived neurotrophic factor also phosphorylated AKT tyrosine residue 315 promoting activation of AKT (28). AKT tyrosine residue 474 was phosphorylated when cells were treated with the tyrosine phosphatase inhibitor pervanadate, and phosphorylation of tyrosine 474 contributed to full activation of AKT (12). Recently, the nonreceptor tyrosine kinase Ack1 was shown to regulate AKT tyrosine phosphorylation and activation (37).Here we show that AKT is a cytoplasmic substrate of the intracellular tyrosine kinase PTK6. We identify the tyrosine residues on AKT that are targeted by PTK6, and we demonstrate that tyrosine phosphorylation plays a role in regulating association between PTK6 and AKT. In addition, we show that PTK6 promotes AKT activation and cell proliferation in a Src-independent manner.     

Extracellular Signal-Regulated Kinase Promotes Rho-Dependent Focal Adhesion Formation by Suppressing p190A RhoGAP

Cell migration is critical for normal development and for pathological processes including cancer cell metastasis. Dynamic remodeling of focal adhesions and the actin cytoskeleton are crucial determinants of cell motility. The Rho family and the mitogen-activated protein kinase (MAPK) module consisting of MEK-extracellular signal-regulated kinase (ERK) are important regulators of these processes, but mechanisms for the integration of these signals during spreading and motility are incompletely understood. Here we show that ERK activity is required for fibronectin-stimulated Rho-GTP loading, Rho-kinase function, and the maturation of focal adhesions in spreading cells. We identify p190A RhoGAP as a major target for ERK signaling in adhesion assembly and identify roles for ERK phosphorylation of the C terminus in p190A localization and activity. These observations reveal a novel role for ERK signaling in adhesion assembly in addition to its established role in adhesion disassembly.Cell migration is a highly coordinated process essential for physiological and pathological processes (69). Signaling through Rho family GTPases (e.g., Rac, Cdc42, and Rho) is crucial for cell migration. Activated Rac and Cdc42 are involved in the production of a dominant lamellipodium and filopodia, respectively, whereas Rho-stimulated contractile forces are required for tail retraction and to maintain adhesion to the matrix (57, 58, 68). Rac- and Cdc42-dependent membrane protrusions are driven by the actin cytoskeleton and the formation of peripheral focal complexes; Rho activation stabilizes protrusions by stimulating the formation of mature focal adhesions and stress fibers. Active Rho influences cytoskeletal dynamics through effectors including the Rho kinases (ROCKs) (2, 3).Rho activity is stimulated by GEFs that promote GTP binding and attenuated by GTPase-activating proteins (GAPs) that enhance Rho''s intrinsic GTPase activity. However, due to the large number of RhoGEFs and RhoGAPs expressed in mammalian cells, the molecular mechanisms responsible for regulation of Rho activity in time and space are incompletely understood. p190A RhoGAP (hereafter p190A) is implicated in adhesion and migration signaling. p190A contains an N-terminal GTPase domain, a large middle domain juxtaposed to the C-terminal GAP domain, and a short C-terminal tail (74). The C-terminal tail of ∼50 amino acids is divergent between p190A and the closely related family member p190B (14) and thus may specify the unique functional roles for p190A and p190B revealed in gene knockout studies (10, 11, 41, 77, 78). p190A activity is dynamically regulated in response to external cues during cell adhesion and migration (5, 6, 59). Arthur et al. (5) reported that p190A activity is required for the transient decrease in RhoGTP levels seen in fibroblasts adhering to fibronectin. p190A activity is positively regulated by tyrosine phosphorylation (4, 5, 8, 17, 31, 39, 40, 42): phosphorylation at Y1105 promotes its association with p120RasGAP and subsequent recruitment to membranes or cytoskeleton (8, 17, 27, 31, 71, 75, 84). However, Y1105 phosphorylation is alone insufficient to activate p190A GAP activity (39). While the functions of p190A can be irreversibly terminated by ubiquitinylation in a cell-cycle-dependent manner (80), less is known about reversible mechanisms that negatively regulate p190A GAP activity during adhesion and motility.The integration of Rho family GTPase and extracellular signal-regulated kinase (ERK) signaling is important for cell motility (48, 50, 63, 76, 79). Several studies have demonstrated a requirement for ERK signaling in the disassembly of focal adhesions in migrating cells, in part through the activation of calpain proteases (36, 37) that can downregulate focal adhesion kinase (FAK) signaling (15), locally suppress Rho activity (52), and sever cytoskeletal linkers to focal adhesions (7, 33). Inhibition of ERK signaling increases focal adhesion size and retards disassembly of focal adhesions in adherent cells (57, 64, 85, 86). It is also recognized that ERK modulates Rho-dependent cellular processes, including membrane protrusion and migration (18, 25, 64, 86). Interestingly, ERK activated in response to acute fibronectin stimulation localizes not only to mature focal adhesions, but also to peripheral focal complexes (32, 76). Since these complexes can either mature or be turned over (12), ERK may play a distinct role in focal adhesion assembly. ERK is proposed to promote focal adhesion formation by activating myosin light chain kinase (MLCK) (21, 32, 50).Here we find that ERK activity is required for Rho activation and focal adhesion formation during adhesion to fibronectin and that p190A is an essential target of ERK signaling in this context. Inspection of the p190A C terminus reveals a number of consensus ERK sites and indeed p190A is phosphorylated by recombinant ERK only on its C terminus in vitro, and on the same C-terminal peptide in vivo. Mutation of the C-terminal ERK phosphorylation sites to alanine increases the biochemical and biological activity of p190A. Finally, inhibition of MEK or mutation of the C-terminal phosphorylation sites enhances retention of p190A in peripheral membranes during spreading on fibronectin. Our data support the conclusion that ERK phosphorylation inhibits p190A allowing increases in RhoGTP and cytoskeletal changes necessary for focal adhesion formation.     

Differences in the Regulatory and Functional Effects of the Us3 Protein Kinase Activities of Herpes Simplex Virus 1 and 2

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases with amino acid sequences that are conserved in the subfamily Alphaherpesvirinae (6, 24, 36). Based on studies showing that recombinant Us3 mutants of HSV-1 and HSV-2 have significantly impaired viral replication and virulence in mice models, it has been concluded that both HSV-1 and HSV-2 Us3 protein kinases play important roles in viral replication and pathogenicity in vivo (25, 33, 41). In contrast, HSV-1 and HSV-2 Us3 protein kinases are not essential for growth in tissue culture cells (33, 36). Thus, recombinant Us3 mutants grow as well as wild-type viruses in Vero cells, and the mutants exhibit modestly impaired replication in HEp-2 cells (33, 36, 39, 40). The possible functions of Us3 have been extensively studied and gradually elucidated for HSV-1 Us3, but much less is known about HSV-2 Us3. These functions include (i) blocking apoptosis (1, 22, 30, 31, 35); (ii) promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (39, 40, 45); (iii) redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (14, 37, 38) and cellular proteins, including lamin A/C and emerin (21, 27, 28); (iv) controlling infected cell morphology (13, 31, 32); and (v) downregulating cell surface expression of viral envelope glycoprotein B (gB) (12).To determine the molecular mechanisms for a viral protein kinase''s effects in infected cells, the kinase''s physiological substrates and its phosphorylation sites must be identified. This can involve studies showing that the altered phenotypes observed in cells infected with a mutant virus lacking the protein kinase activity is also detected in cells infected with a mutant virus in which the substrate''s phosphorylation sites have been modified by mutations. Although more than 15 potential HSV Us3 substrates have been reported, HSV-1 Us3 phosphorylation of only three substrates (Us3 itself, gB, and UL31) has been demonstrated to be linked directly with Us3 functions in infected cells (12, 13, 29, 41) as follows. (i) Us3 has been reported to autophosphorylate serine at position 147 (Ser-147), and this phosphorylation augments Us3''s kinase activity in infected cells (13, 41). Even though only a small fraction of Us3 is autophosphorylated at Ser-147 in infected cells, alanine replacement of Ser-147 in Us3 significantly reduced HSV-1 replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (41). These results indicated that Us3 kinase activity was, in part, regulated by autophosphorylation of Ser-147, and regulation of Us3 activity by autophosphorylation played a critical role in viral replication in vivo and HSV-1 pathogenesis. (ii) It has been reported that HSV-1 Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, and this phosphorylation downregulates the cell surface expression of gB (12). Us3 phosphorylation of gB at Thr-887 also has been proposed to be involved in the regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with mutant viruses carrying the amino acid substitution mutation T887A in gB and lacking the capacity to produce gH (45). The Us3 phosphorylation of gB at Thr-887 appeared to be critical for HSV-1 replication and pathogenesis in vivo, based on studies showing that the T887A substitution in the phosphorylation site in gB significantly reduced viral replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (Takahiko Imai, Ken Sagou, and Yasushi Kawaguchi, unpublished observations). (iii) It has been shown that Us3 phosphorylated some or all of the six serines in the UL31 N-terminal region, and this phosphorylation regulated the proper localization of UL31 and UL34 at the nuclear membrane and nuclear egress of nucleocapsids (29). Thus, the molecular basis of HSV-1 Us3 effects in infected cells have been gradually elucidated.However, the Us3 phosphorylation sites in Us3 itself and in gB are not conserved between HSV serotypes (12, 13). The amino acid residues in HSV-2 Us3 and gB corresponding to HSV-1 Us3 Ser-147 and gB Thr-887 are aspartic acid (Asp-147) and alanine (Ala-887), respectively. These results suggest that some HSV-1 Us3 functions, such as regulation of its own catalytic activity and control of gB expression on the cell surface, are not regulated by HSV-2 Us3 or are regulated in a manner(s) different from HSV-1 Us3. In agreement with this suggestion, there is a marked difference between HSV-1 and HSV-2 virulence in mice following intracerebral infection, with the HSV-1 Us3 null mutant being >10⁴-fold less virulent than the parent wild-type virus (25), while the HSV-2 Us3 null mutant was only ∼10-fold less virulent (33). Although these results were from different reports and the mouse strains used in the studies were different, they indicate that some HSV-1 Us3 functions are different from those of HSV-2 Us3.Therefore, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases. It was of particular interest to examine whether Asp-147 in HSV-2 Us3 is required for its own kinase activity, since it is well established that acidic amino acids such as Asp or glutamic acid sometimes mimic the negative charges produced by phosphorylation (29, 46). In the present study, using a genetic manipulation system of HSV-2 with our newly constructed HSV-2 bacterial artificial chromosome (BAC) clone, we have shown that HSV-2 Us3 exhibited marked differences from HSV-1 Us3 in its catalytic functions, including the regulation of UL31/UL34 localization, nuclear egress of nucleocapsids, cell surface expression of gB, and virulence in mice. We also found that Asp-147 in HSV-2 Us3 was critical for its kinase activity, raising a possibility that the activity of Us3 kinases was regulated differently in HSV-1 and HSV-2.     

Functional and Complementary Phosphorylation State Attributes of Human Insulin-like Growth Factor-Binding Protein-1 (IGFBP-1) Isoforms Resolved by Free Flow Electrophoresis

Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size. High concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) have been associated with FGR. Phosphorylation of IGFBP-1 is a mechanism by which insulin-like growth factor-I (IGF-I) bioavailability can be modulated in FGR. In this study a novel strategy was designed to determine a link between IGF-I affinity and the concomitant phosphorylation state characteristics of IGFBP-1 phosphoisoforms. Using free flow electrophoresis (FFE), multiple IGFBP-1 phosphoisoforms in amniotic fluid were resolved within pH 4.43–5.09. The binding of IGFBP-1 for IGF-I in each FFE fraction was determined with BIAcore biosensor analysis. The IGF-I affinity (K) for different IGFBP-1 isoforms ranged between 1.12e−08 and 4.59e−07. LC-MS/MS characterization revealed four phosphorylation sites, Ser(P)⁹⁸, Ser(P)¹⁰¹, Ser(P)¹¹⁹, and Ser(P)¹⁶⁹, of which Ser(P)⁹⁸ was new. Although the IGF-I binding affinity for IGFBP-1 phosphoisoforms across the FFE fractions did not correlate with phosphopeptide intensities for Ser(P)¹⁰¹, Ser(P)⁹⁸, and Ser(P)¹⁶⁹ sites, a clear association was recorded with Ser(P)¹¹⁹. Our data demonstrate that phosphorylation at Ser¹¹⁹ plays a significant role in modulating affinity of IGFBP-1 for IGF-I. In addition, an altered profile of IGFBP-1 phosphoisoforms was revealed between FGR and healthy pregnancies that may result from potential site-specific phosphorylation. This study provides a strong basis for use of this novel approach in establishing the linkage between phosphorylation of IGFBP-1 and FGR. This overall strategy will also be broadly applicable to other phosphoproteins with clinical and functional significance.The insulin-like growth factor (IGF)¹ axis plays an important role in human fetal growth and development. Insulin-like growth factor-binding protein-1 (IGFBP-1) is a major IGF-binding protein in amniotic fluid (AF) (1, 2). The physiological role of IGFBP-1 is considered to be highly dependent on its differential phosphorylation (3–5). Phosphorylation of IGFBP-1 increases its affinity for IGF-I (6), suggesting that IGFBP-1 may modulate the action of IGF-I specifically with respect to fetal and placental growth (4, 7).AF is a dynamic and complex biofluid and reflects the physiological status of the developing fetus (8). Fetal growth restriction (FGR) is a condition in which a fetus is unable to achieve its genetically determined potential size. The concentration of total IGFBP-1 is increased in FGR (9–12). Multiple phosphorylated species of IGFBP-1 have been detected during healthy pregnancy in both maternal circulation and in AF throughout gestation (1, 13, 14). Several studies have considered the clinical implications of IGFBP-1 phosphorylation, focusing on correlating variable ratios of high to low concentrations of IGFBP-1 phosphoisoforms with fetal outcome in FGR pregnancies (15–19). Although phosphorylation of IGFBP-1 has since been suggested to be critical, the predictive or functional value of IGFBP-1 phosphorylation in FGR is still not clear. The inconsistency in measurements of variable degrees of IGFBP-1 phosphorylation by ELISAs has resulted in inconclusive findings (20).IGFBP-1 phosphoisoforms have been characterized previously using conventional methods (1, 13, 14). IGFBP-1, although relatively abundant in AF, still represents less than 0.01% of the total protein content (21). With restricted volumes available from clinical samples, isolation of functional IGFBP-1 phosphoisoforms using traditional approaches (13, 22, 23) is challenging. Our goal is to obtain a comprehensive understanding of the clinical and functional implications of IGFBP-1 phosphorylation in human FGR pregnancies. We developed an efficient, reproducible, and entirely liquid-based native IEF separation technology based on free flow electrophoresis (FFE) (24) to facilitate characterization of both the state of phosphorylation and IGF-I binding kinetics of variably phosphorylated IGFBP-1 isoforms in a clinical sample. As a prerequisite to application of this approach clinically, we also evaluated representative AF samples to determine whether or not differential patterns of IGFBP-1 phosphorylation exist in FGR and whether these changes could be attributed to augmentation of IGF-I affinity in the disease.     

Trafficking of UL37 Proteins into Mitochondrion-Associated Membranes during Permissive Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) UL37 proteins traffic sequentially from the endoplasmic reticulum (ER) to the mitochondria. In transiently transfected cells, UL37 proteins traffic into the mitochondrion-associated membranes (MAM), the site of contact between the ER and mitochondria. In HCMV-infected cells, the predominant UL37 exon 1 protein, pUL37x1, trafficked into the ER, the MAM, and the mitochondria. Surprisingly, a component of the MAM calcium signaling junction complex, cytosolic Grp75, was increasingly enriched in heavy MAM from HCMV-infected cells. These studies show the first documented case of a herpesvirus protein, HCMV pUL37x1, trafficking into the MAM during permissive infection and HCMV-induced alteration of the MAM protein composition.The human cytomegalovirus (HCMV) UL37 immediate early (IE) locus expresses multiple products, including the predominant UL37 exon 1 protein, pUL37x1, also known as viral mitochondrion-localized inhibitor of apoptosis (vMIA), during lytic infection (16, 22, 24, 39, 44). The UL37 glycoprotein (gpUL37) shares UL37x1 sequences and is internally cleaved, generating pUL37_NH2 and gpUL37_COOH (2, 22, 25, 26). pUL37x1 is essential for the growth of HCMV in humans (17) and for the growth of primary HCMV strains (20) and strain AD169 (14, 35, 39, 49) but not strain TownevarATCC in permissive human fibroblasts (HFFs) (27).pUL37x1 induces calcium (Ca²⁺) efflux from the endoplasmic reticulum (ER) (39), regulates viral early gene expression (5, 10), disrupts F-actin (34, 39), recruits and inactivates Bax at the mitochondrial outer membrane (MOM) (4, 31-33), and inhibits mitochondrial serine protease at late times of infection (28).Intriguingly, HCMV UL37 proteins localize dually in the ER and in the mitochondria (2, 9, 16, 17, 24-26). In contrast to other characterized, similarly localized proteins (3, 6, 11, 23, 30, 38), dual-trafficking UL37 proteins are noncompetitive and sequential, as an uncleaved gpUL37 mutant protein is ER translocated, N-glycosylated, and then imported into the mitochondria (24, 26).Ninety-nine percent of ∼1,000 mitochondrial proteins are synthesized in the cytosol and directly imported into the mitochondria (13). However, the mitochondrial import of ER-synthesized proteins is poorly understood. One potential pathway is the use of the mitochondrion-associated membrane (MAM) as a transfer waypoint. The MAM is a specialized ER subdomain enriched in lipid-synthetic enzymes, lipid-associated proteins, such as sigma-1 receptor, and chaperones (18, 45). The MAM, the site of contact between the ER and the mitochondria, permits the translocation of membrane-bound lipids, including ceramide, between the two organelles (40). The MAM also provides enriched Ca²⁺ microdomains for mitochondrial signaling (15, 36, 37, 43, 48). One macromolecular MAM complex involved in efficient ER-to-mitochondrion Ca²⁺ transfer is comprised of ER-bound inositol 1,4,5-triphosphate receptor 3 (IP3R3), cytosolic Grp75, and a MOM-localized voltage-dependent anion channel (VDAC) (42). Another MAM-stabilizing protein complex utilizes mitofusin 2 (Mfn2) to tether ER and mitochondrial organelles together (12).HCMV UL37 proteins traffic into the MAM of transiently transfected HFFs and HeLa cells, directed by their NH₂-terminal leaders (8, 47). To determine whether the MAM is targeted by UL37 proteins during infection, we fractionated HCMV-infected cells and examined pUL37x1 trafficking in microsomes, mitochondria, and the MAM throughout all temporal phases of infection. Because MAM domains physically bridge two organelles, multiple markers were employed to verify the purity and identity of the fractions (7, 8, 19, 46, 47).(These studies were performed in part by Chad Williamson in partial fulfillment of his doctoral studies in the Biochemistry and Molecular Genetics Program at George Washington Institute of Biomedical Sciences.)HFFs and life-extended (LE)-HFFs were grown and not infected or infected with HCMV (strain AD169) at a multiplicity of 3 PFU/cell as previously described (8, 26, 47). Heavy (6,300 × g) and light (100,000 × g) MAM fractions, mitochondria, and microsomes were isolated at various times of infection and quantified as described previously (7, 8, 47). Ten- or 20-μg amounts of total lysate or of subcellular fractions were resolved by SDS-PAGE in 4 to 12% Bis-Tris NuPage gels (Invitrogen) and examined by Western analyses (7, 8, 26). Twenty-microgram amounts of the fractions were not treated or treated with proteinase K (3 μg) for 20 min on ice, resolved by SDS-PAGE, and probed by Western analysis. The blots were probed with rabbit anti-UL37x1 antiserum (DC35), goat anti-dolichyl phosphate mannose synthase 1 (DPM1), goat anti-COX2 (both from Santa Cruz Biotechnology), mouse anti-Grp75 (StressGen Biotechnologies), and the corresponding horseradish peroxidase-conjugated secondary antibodies (8, 47). Reactive proteins were detected by enhanced chemiluminescence (ECL) reagents (Pierce), and images were digitized as described previously (26, 47).     

Site-Specific mTOR Phosphorylation Promotes mTORC1-Mediated Signaling and Cell Growth

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) functions as a rapamycin-sensitive environmental sensor that promotes cellular biosynthetic processes in response to growth factors and nutrients. While diverse physiological stimuli modulate mTORC1 signaling, the direct biochemical mechanisms underlying mTORC1 regulation remain poorly defined. Indeed, while three mTOR phosphorylation sites have been reported, a functional role for site-specific mTOR phosphorylation has not been demonstrated. Here we identify a new site of mTOR phosphorylation (S1261) by tandem mass spectrometry and demonstrate that insulin-phosphatidylinositol 3-kinase signaling promotes mTOR S1261 phosphorylation in both mTORC1 and mTORC2. Here we focus on mTORC1 and show that TSC/Rheb signaling promotes mTOR S1261 phosphorylation in an amino acid-dependent, rapamycin-insensitive, and autophosphorylation-independent manner. Our data reveal a functional role for mTOR S1261 phosphorylation in mTORC1 action, as S1261 phosphorylation promotes mTORC1-mediated substrate phosphorylation (e.g., p70 ribosomal protein S6 kinase 1 [S6K1] and eukaryotic initiation factor 4E binding protein 1) and cell growth to increased cell size. Moreover, Rheb-driven mTOR S2481 autophosphorylation and S6K1 phosphorylation require S1261 phosphorylation. These data provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function and suggest a model whereby insulin-stimulated mTOR S1261 phosphorylation promotes mTORC1 autokinase activity, substrate phosphorylation, and cell growth.The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine/threonine protein kinase, senses and integrates signals from diverse environmental cues (14, 31, 50, 74). mTOR associates with different partner proteins to form functionally distinct signaling complexes (4). The immunosuppressive drug rapamycin acutely inhibits signaling by mTOR complex 1 (mTORC1) (22), which contains mTOR, mLST8/GβL, raptor, and PRAS40 (24, 33, 34, 54, 67). Rapamycin fails to acutely inhibit signaling by mTORC2, which contains mTOR, mLST8/GβL, rictor, mSin1, and PRR5/Protor (18, 32, 47, 55, 73, 76). mTORC1 promotes various biosynthetic processes, including protein synthesis, cell growth (an increase in cell mass and size), and cell proliferation (an increase in cell number) (14, 40, 74). During growth factor (e.g., insulin) and nutrient (e.g., amino acids and glucose) sufficiency, mTORC1 phosphorylates the translational regulators p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1) to coordinately upregulate protein biosynthesis (40). Both S6K1 and 4EBP1 contain a TOR signaling motif, which mediates their interaction with raptor and thus facilitates their recruitment to the mTOR kinase (10, 44, 57, 58). In addition to regulating protein synthesis, mTORC1-mediated phosphorylation of S6K1 and 4EBP also promotes cell growth and cell cycle progression (15, 16). While more recently identified and thus less well characterized than mTORC1, mTORC2 mediates the phosphorylation of AGC kinase family members (e.g., Akt [also known as protein kinase B, PKB], PKCα, and SGK1) on their hydrophobic motifs and modulates the organization of the actin cytoskeleton (20, 26, 32, 55, 56).The insulin pathway represents the best-characterized activator of mTORC1 signaling to date, and thus many signaling intermediates that link insulin receptor activation to mTORC1 have been identified (12, 31). Complementary work using Drosophila melanogaster genetics and mammalian cell culture identified TSC1 (hamartin) and TSC2 (tuberin) as upstream negative regulators of mTORC1 (27). Inactivation of either the TSC1 or TSC2 genes, whose protein products heterodimerize to form a tumor suppressor complex, causes the development of benign tumors in diverse organs in both humans and rodents, a disease known as tuberous sclerosis complex (TSC) (36). TSC2 contains a GTPase-activating protein domain that acts on Rheb, a Ras-like GTP binding protein that activates mTORC1 (27). Thus, in TSC-deficient cells, constitutive Rheb-GTP leads to chronically high mTORC1 signaling. While the mechanism by which Rheb-GTP activates mTORC1 remains incompletely understood, Rheb coimmunoprecipitates with mTOR and directly activates mTORC1 kinase activity in vivo and in vitro when GTP bound (2, 38, 54). Rheb has been reported to augment the activity of PLD1, an enzyme that catalyzes the production of the lipid second messenger phosphatidic acid, which contributes to the mitogenic activation of mTORC1 signaling (13, 62). Additionally, Rheb-GTP was reported to induce the dissociation of the endogenous mTOR inhibitor FKBP38 (3), although aspects of this model have been questioned (72). Insulin/phosphatidylinositol 3-kinase (PI3K) signaling reduces the inhibitory effect of TSC on mTORC1 via Akt-mediated phosphorylation of TSC2 (29, 42, 64). Additionally, Ras-regulated signaling via mitogen-activated protein kinase (MAPK) and RSK also inhibits TSC via PI3K/Akt-independent phosphorylation of TSC2 (39, 51, 63). In contrast, glucose deprivation enhances TSC''s inhibitory effect on mTORC1 signaling via AMP-activated protein kinase (AMPK)-mediated phosphorylation of TSC2 (on different sites) (30). Thus, TSC functions as a central nexus of diverse physiological signals to fine-tune mTORC1 signaling depending on environmental conditions (27). While the mechanism by which amino acids promote mTORC1 signaling has remained elusive, compelling new data reveal that the Rag GTPases link amino acid sensing to mTORC1 activation (35, 52, 53). During amino acid sufficiency, GTP-bound Rag heterodimers bind raptor and recruit mTORC1 to an endomembrane compartment that contains the mTORC1 activator Rheb; thus, amino acid sufficiency may function to prime mTORC1 for subsequent growth factor-mediated activation via a dynamic subcellular redistribution mechanism (52).Despite the well-characterized regulation of mTORC1 signaling by growth factors (e.g., insulin), nutrients (e.g., amino acids and glucose), and cellular stress (e.g., hypoxia) and the identification of numerous signaling mediators of these pathways, the direct molecular mechanisms by which cellular signals modulate mTORC1 action remain obscure (31). While three phosphorylation sites (P-sites) on mTOR have been reported to date (T2446, S2448, and S2481), no function has yet been ascribed to any site (7, 43, 49, 59). Here we identify S1261 as a novel mTOR phosphorylation site in vivo in cultured mammalian cells and provide the first evidence that site-specific mTOR phosphorylation regulates mTORC1 function. We show that insulin signals via the PI3K/TSC/Rheb pathway in an amino acid-dependent and rapamycin-insensitive manner to promote mTOR S1261 phosphorylation, which regulates mTORC1 autokinase activity, biochemical signaling to downstream substrates, and cell growth to increased cell size, a major cellular function of mTORC1. Elucidation of the molecular mechanisms underlying mTORC1 regulation will enable us to better understand how mTORC1 senses environmental stimuli to control cellular physiology. As aberrantly upregulated mTORC1 signaling likely contributes to cancer, insulin-resistant diabetes, and cardiovascular diseases, understanding mTORC1 regulation may aid in the development of novel therapeutics for these prevalent human diseases (11, 21, 28).     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

Coinfecting Prion Strains Compete for a Limiting Cellular Resource

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP^Sc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP^Sc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP^Sc does not convert all of the available PrP^C to PrP^Sc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP^C and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP^Sc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrP^Sc, a misfolded isoform of the noninfectious host encoded protein PrP^C (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrP^Sc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrP^Sc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.