Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ��

A key set of reactions for the initiation of new DNA strands during herpes simplex virus-1 replication consists of the primase-catalyzed synthesis of short RNA primers followed by polymerase-catalyzed DNA synthesis (i.e. primase-coupled polymerase activity). Herpes primase (UL5-UL52-UL8) synthesizes products from 2 to ∼13 nucleotides long. However, the herpes polymerase (UL30 or UL30-UL42) only elongates those at least 8 nucleotides long. Surprisingly, coupled activity was remarkably inefficient, even considering only those primers at least 8 nucleotides long, and herpes polymerase typically elongated <2% of the primase-synthesized primers. Of those primers elongated, only 4–26% of the primers were passed directly from the primase to the polymerase (UL30-UL42) without dissociating into solution. Comparing RNA primer-templates and DNA primer-templates of identical sequence showed that herpes polymerase greatly preferred to elongate the DNA primer by 650–26,000-fold, thus accounting for the extremely low efficiency with which herpes polymerase elongated primase-synthesized primers. Curiously, one of the DNA polymerases of the host cell, polymerase α (p70-p180 or p49-p58-p70-p180 complex), extended herpes primase-synthesized RNA primers much more efficiently than the viral polymerase, raising the possibility that the viral polymerase may not be the only one involved in herpes DNA replication.Herpes simplex virus 1 (HSV-1)² encodes seven proteins essential for replicating its double-stranded DNA genome; five of these encode the heterotrimeric helicase-primase (UL5-UL52-UL8 gene products) and the heterodimeric polymerase (UL30-UL42 gene products) (1, 2). The helicase-primase unwinds the DNA at the replication fork and generates single-stranded DNA for both leading and lagging strand synthesis. Primase synthesizes short RNA primers on the lagging strand that the polymerase presumably elongates using dNTPs (i.e. primase-coupled polymerase activity). These two protein complexes are thought to replicate the viral genome on both the leading and lagging strands (1, 2).Previous studies have focused on the helicase-primase and polymerase separately. The helicase-primase contains three subunits, UL5, UL52, and UL8 in a 1:1:1 ratio (3–5). The UL5 subunit has helicase-like motifs and the UL52 subunit has primase-like motifs, yet the minimal active complex that demonstrates either helicase or primase activities contains both UL5 and UL52 (6, 7). Although the UL8 subunit has no known catalytic activity, several functions have been proposed, including enhancing helicase and primase activities, enhancing primer synthesis on ICP8 (the HSV-1 single-stranded binding protein)-coated DNA strands, and facilitating formation of the replisome (8–12). Although primase will synthesize short (2–3 nucleotides long) primers on a variety of template sequences, synthesis of longer primers up to 13 nucleotides long requires the template sequence, 3′-deoxyguanidine-pyrimidine-pyrimidine-5′ (13). Primase initiates synthesis at the first pyrimidine via the polymerization of two purine NTPs (13). Even after initiation at this sequence, however, the vast majority of products are only 2–3 nucleotides long (13, 14).The herpes polymerase consists of the UL30 subunit, which has polymerase and 3′ → 5′ exonuclease activities (1, 2), and the UL42 subunit, which serves as a processivity factor (15–17). Unlike most processivity factors that encircle the DNA, the UL42 protein binds double-stranded DNA and thus directly tethers the polymerase to the DNA (18). Using pre-existing DNA primer-templates as the substrate, the heterodimeric polymerase (UL30-UL42) incorporates dNTPs at a rate of 150 s^–1, a rate much faster than primer synthesis (for primers >7 nucleotides long, 0.0002–0.01 s^–1) (19, 20).We examined primase-coupled polymerase activity by the herpes primase and polymerase complexes. Although herpes primase synthesizes RNA primers 2–13 nucleotides long, the polymerase only effectively elongates those at least 8 nucleotides long. Surprisingly, the polymerase elongated only a small fraction of the primase-synthesized primers (<1–2%), likely because of the polymerase elongating RNA primer-templates much less efficiently than DNA primer-templates. In contrast, human DNA polymerase α (pol α) elongated the herpes primase-synthesized primers very efficiently. The biological significance of these data is discussed.     

Sequential Loading of Saccharomyces cerevisiae Ku and Cdc13p to Telomeres

Ku is a heterodimeric protein involved in nonhomologous end-joining of the DNA double-stranded break repair pathway. It binds to the double-stranded DNA ends and then activates a series of repair enzymes that join the broken DNA. In addition to its function in DNA repair, the yeast Saccharomyces cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes that affect telomere function. The yeast telomeres are composed of duplex C_1–3(A/T)G_1–3 telomeric DNA repeats plus single-stranded TG_1–3 telomeric DNA tails. Here we show that Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that mimics the structure of telomeres. Addition of Cdc13p, a single-stranded telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of a ternary complex with Cdc13p binding to the single-stranded tail of the DNA substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail inhibits the binding of Yku, the results suggested that loading of Yku and Cdc13p to telomeres is sequential. Through generating a double-stranded break near telomeric DNA sequences, we found that Ku protein appears to bind to the de novo synthesized telomeres earlier than that of Cdc13p in vivo. Thus, our results indicated that Yku interacts directly with telomeres and that sequential loading of Yku followed by Cdc13p to telomeres is required for both proteins to form a ternary complex on telomeres. Our results also offer a mechanism that the binding of Cdc13p to telomeres might prevent Yku from initiating DNA double-stranded break repair pathway on telomeres.DNA damages in the form of double-stranded breaks (DSBs)⁴ compromise the integrity of genomes. Failure in repairing or mis-repairing double-stranded breaks can lead to chromosome instability and eventually cell death or cancer (1). Double-stranded breaks are repaired by two main pathways, the homologous recombination and nonhomologous DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to bind to the DNA ends to initiate the repair pathway (2). Upon binding, Ku then recruits a series of repair enzymes to join the broken ends (2). Ku is a heterodimeric protein composed of 70- and ∼80-kDa subunits. In Saccharomyces cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical configuration of the broken ends could be very diverse on DSBs, Ku binds to double-stranded ends in a sequence- and energy-independent manner. It is capable of binding to DNA ends with blunt 3′-overhangs or 5′-overhangs as well as double-stranded DNA with nicks, gaps, or internal loops (3–7). However, Ku does not have high affinity to single-stranded DNA. The crystal structure of human Ku heterodimer indicates that it forms a ring structure that encircles duplex DNA (7). This unique structure feature enables Ku to recognize DNA ends and achieves its high affinity binding.In additional to the role in double-stranded break repair, Ku was shown to be a component of telomeric protein-DNA complex in yeast and mammals (8–10). Telomeres are terminal structures of chromosomes composed of short tandem repeated sequences (11, 12). Mutation of YKU70 or YKU80 causes defects in telomere structure (13–15), telomere silencing (16–19), and replication timing of telomeres (20). The function of yeast Ku (Yku) on telomeres could mediate through protein-protein interaction with Sir4p or protein-RNA interaction with Tlc1 RNA (21, 22). For example, through the interaction with Sir4p, Yku selectively affects telomeres silencing but not the silent mating type loci (17). Yku could also bind to telomerase Tlc1 RNA for telomere length maintenance (22). Judged by the DNA binding activity of Yku, it is reasonable to suggest that it may bind directly to telomeric DNA. Indeed, it was shown that human Ku is capable of binding directly to telomeric DNA in vitro (15). Moreover, because the deletion of SIR4 in budding yeast (23) or Taz1 in fission yeast (24) does not abolish the association of Ku with chromosomal ends, this suggests that Ku might bind directly to telomeric DNA in cells. However, because yeast telomeres have a short 12–14-mer single-stranded tail (25), it is uncertain whether Yku could pass the single-stranded region to reach its binding site. The direct binding of Yku to telomeric DNA has not been experimentally determined.In contrast to double-stranded breaks, the ends of linear chromosomes are not recognized by repair enzymes as DNA damage. In S. cerevisiae, Cdc13p is the single-stranded TG_1–3 DNA-binding protein that enables cells to differentiate whether the ends of a linear DNA are telomeres or broken ends (26–29). Thus, although the mechanism of how cells prevent the activation of DSB repair pathway in telomere is unclear, it is likely that binding of Cdc13p to telomeres might inhibit the initiation of DNA damage response by the Ku protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA sequences to mimic telomere structure, we showed that Yku binds directly to this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our results also showed that Yku loaded to a de novo synthesized telomere earlier than Cdc13p in vivo. These results support the direct binding of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block the activation of DSB repair pathway on telomeres.     

Long Patch Base Excision Repair Proceeds via Coordinated Stimulation of the Multienzyme DNA Repair Complex

Base excision repair, a major repair pathway in mammalian cells, is responsible for correcting DNA base damage and maintaining genomic integrity. Recent reports show that the Rad9-Rad1-Hus1 complex (9-1-1) stimulates enzymes proposed to perform a long patch-base excision repair sub-pathway (LP-BER), including DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (pol β), flap endonuclease 1 (FEN1), and DNA ligase I (LigI). However, 9-1-1 was found to produce minimal stimulation of FEN1 and LigI in the context of a complete reconstitution of LP-BER. We show here that pol β is a robust stimulator of FEN1 and a moderate stimulator of LigI. Apparently, there is a maximum possible stimulation of these two proteins such that after responding to pol β or another protein in the repair complex, only a small additional response to 9-1-1 is allowed. The 9-1-1 sliding clamp structure must serve primarily to coordinate enzyme actions rather than enhancing rate. Significantly, stimulation by the polymerase involves interaction of primer terminus-bound pol β with FEN1 and LigI. This observation provides compelling evidence that the proposed LP-BER pathway is actually employed in cells. Moreover, this pathway has been proposed to function by sequential enzyme actions in a “hit and run” mechanism. Our results imply that this mechanism is still carried out, but in the context of a multienzyme complex that remains structurally intact during the repair process.The mammalian genome experiences constant stress from both external and internal factors that causes genomic instability. Eukaryotic cells have developed a number of DNA repair pathways that correct DNA damage before it results in permanent chromosomal alteration. Base excision repair (BER)³ is the major pathway responsible for reversing DNA damage sustained by individual nucleotide bases. Mammalian BER is initiated by DNA glycosylases, which recognize structural alteration of a nitrogenous base and excise it leaving an intact sugar-phosphate backbone with an apurinic/apyrimidinic (AP) site (1). AP sites in humans are detected by AP endonuclease 1 (APE1) that cleaves the phosphate backbone of the damaged strand, leaving a nick with a 3′-OH group and a 5′-deoxyribose phosphate (dRP) residue. The dRP-bordered nick is not a substrate for ligation. If the dRP residue is not oxidized or reduced, repair can proceed via a short patch-BER pathway, in which the dRP residue is removed by the 5′-lyase activity of DNA polymerase β (pol β), which concurrently fills in the 1-nt gap, and the resulting nick is sealed by the DNA ligase III-XRCC1 complex (2-4).However, if the oxidative state of the dRP is altered, the lyase activity of pol β is inhibited, but the polymerase activity of pol β can still displace the oxidized or reduced dRP residue into a 2-10-nt 5′ flap intermediate, which will then be cleaved by FEN1 and subsequently joined by LigI (4-7). This process is known as long patch-base excision repair (LP-BER). Recent studies examining the relevance of the two different pathways in vitro predict a predominant role for short patch-BER in the cell as compared with LP-BER (8). Because the cell undergoes constant repair of damaged bases, it is very difficult to assess the relative use of one pathway over the other in vivo. Studies using plasmid DNA containing defined DNA damage have been used as an indirect approach to evaluate the role of the two different BER pathways in cells and the size of the DNA repair patches (9). Results from these studies have shown that repair patches of 6-12 nucleotides are generated during repair of plasmids that contain a single base lesion, at least supporting the existence of LP-BER in vivo.LP-BER has also been proposed to proceed by either a PCNA-dependent sub-pathway involving the use of DNA pol δ/ε or a PCNA-independent sub-pathway that uses only DNA pol β. However, most LP-BER reconstitution experiments in vitro indicate that pol β works more efficiently than pol δ with the other proposed LP-BER proteins. FEN1 is known to stimulate pol β-mediated DNA synthesis on an LP-BER substrate suggesting that these two proteins interact functionally and mechanistically (10). pol β has also been shown to interact with LigI by co-immunoprecipitation experiments indicating that they might be a part of a multiprotein DNA repair complex (11).The heterotrimeric protein complex, Rad9, Rad1, and Hus1 (the 9-1-1 complex), plays a significant role in the early recognition of DNA damage and recruiting appropriate proteins to repair sites. The 9-1-1 complex interacts with several of the proteins involved in the proposed BER pathways, including DNA glycosylases (12-14), APE1 (15), pol β (16), FEN1 (17,18), and LigI (19, 20). In a recent report (15), the 9-1-1 complex was shown to interact both physically and functionally with APE1 and pol β and to stimulate their respective activities. Stimulation of the endonuclease ensures the abasic site is recognized and cleaved off efficiently. Stimulation of nucleotide addition by pol β is expected to promote the LP-BER sub-pathway, as 9-1-1 stimulates the strand displacement activity of pol β, thereby requiring FEN1 flap cleavage before ligation to repair the site of damage. Because 9-1-1 is structurally similar to the sliding clamp PCNA, early studies were focused on determining the effects of 9-1-1 on DNA replication and repair proteins previously shown to be stimulated by PCNA. The 9-1-1 complex has been reported to stimulate both FEN1 cleavage (17, 18) and nick sealing by LigI (20) in vitro. However, the 9-1-1 clamp poorly stimulated FEN1 and LigI in the entire LP-BER-reconstituted system as compared with strong stimulation by 9-1-1 of individual cognate substrates (15). The authors (15) suggest that FEN1 and LigI evolved to respond to stimulation by PCNA and not 9-1-1 during LP-BER. The issue with this explanation is that it does not take into consideration how LP-BER would be efficiently carried out when damage-induced p21 binds and inhibits PCNA (21).To define how 9-1-1 interacts with the components of BER, we have reconstituted the entire LP-BER pathway using purified human enzymes and substrates that simulate an abasic site created after recognition and cleavage of damaged base by a glycosylase. Similar to results of Gembka et al. (15), we observe much less stimulation of either FEN1 or LigI by 9-1-1 in the fully reconstituted system compared with 9-1-1 stimulation of FEN1 on a flap substrate or LigI on a nicked substrate alone. Our subsequent analysis of the protein-protein interactions among the various LP-BER enzymes provides insight into why the 9-1-1 clamp exhibits minimal stimulation in the reconstituted system. Moreover, our mechanistic characterization of the significant role of pol β in mediating the activities of various enzymes in the multiprotein repair complex both explains the behavior of 9-1-1 and strongly suggests the existence of the LP-BER pathway in vivo.     

Sequential protein kinase C (PKC)-dependent and PKC-independent protein kinase D catalytic activation via Gq-coupled receptors: differential regulation of activation loop Ser(744) and Ser(748) phosphorylation

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸ revealed striking PKC-independent phosphorylation of Ser⁷⁴⁸ as well as Ser⁷⁴⁴ phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors (1). Many of these agonists bind to G protein-coupled receptors (GPCRs),⁴ activate heterotrimeric G proteins and stimulate isoforms of the phospholipase C family, including β, γ, δ, and ε (reviewed in Refs. 1 and 2). Activated phospholipase Cs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate mobilizes Ca²⁺ from intracellular stores (3, 4) whereas DAG directly activates the classic (α, β, and γ) and novel (δ, ε, η, and θ) isoforms of PKC (5–7). Although it is increasingly recognized that each PKC isozyme has specific functions in vivo (5–8), the mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely defined.PKD, also known initially as PKCμ (9, 10), and two recently identified serine protein kinases termed PKD2 (11) and PKCν/PKD3 (12, 13), which are similar in overall structure and primary amino acid sequence to PKD (14), constitute a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase group (15) and separate from the previously identified PKCs (14). Salient features of PKD structure include an N-terminal regulatory region containing a tandem repeat of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain) that confers high affinity binding to phorbol esters and DAG (9, 16, 17), followed by a pleckstrin homology (PH) domain that negatively regulates catalytic activity (18, 19). The C-terminal region of the PKDs contains its catalytic domain, which is distantly related to Ca²⁺-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity maintained by the N-terminal domain, which represses the catalytic activity of the enzyme by autoinhibition. Consistent with this model, deletions or single amino acid substitutions in the PH domain result in constitutive kinase activity (18–20). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (21). In response to cellular stimuli, PKD is converted from a low activity form into a persistently active form that is retained during isolation from cells, as shown by in vitro kinase assays performed in the absence of lipid co-activators (21, 22). PKD activation has been demonstrated in response to engagement of specific GPCRs either by regulatory peptides (23–30) or lysophosphatidic acid (27, 31, 32); signaling through G_q, G₁₂, G_i, and Rho (27, 31–34); activation of receptor tyrosine kinases, such as the platelet-derived growth factor receptor (23, 35, 36); cross-linking of B-cell receptor and T-cell receptor in B and T lymphocytes, respectively (37–40); and oxidative stress (41–44).Throughout these studies, multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF 109203X or Gö 6983) that do not directly inhibit PKD catalytic activity (21, 22), implying that PKD activation in intact cells is mediated, directly or indirectly, through PKCs. In line with this conclusion, cotransfection of PKD with active mutant forms of “novel” PKCs (PKCs δ, ε, η, and θ) resulted in robust PKD activation in the absence of cell stimulation (21, 44–46). Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade in response to multiple GPCR agonists in a broad range of cell types, including normal and cancer cells (reviewed in Ref. 14). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as the activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (reviewed in Ref. 14). Collectively, these findings demonstrated the existence of rapidly activated PKC-PKD protein kinase cascade(s) and raised the possibility that some PKC-dependent biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular activities and processes, including signal transduction (30, 47–49), chromatin modification (50), Golgi organization and function (51, 52), c-Jun function (47, 53, 54), NFκB-mediated gene expression (43, 55, 56), and cell survival, migration, and differentiation and DNA synthesis and proliferation (reviewed in Ref. 14). Thus, mounting evidence indicates that PKD has a remarkable diversity of both its signal generation and distribution and its potential for complex regulatory interactions with multiple downstream pathways, leading to multiple responses, including long term cellular events. Despite increasing recognition of its importance, very little is known about the mechanism(s) of sustained PKD activation as opposed to the well documented rapid, PKC-dependent PKD activation.The results presented here demonstrate that prolonged GPCR-induced PKD activation is mediated by sequential PKC-dependent and PKC-independent phases of regulation. We report here, for the first time, that PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies expand previous models of PKD regulation by identifying a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different G_q-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸. Transphosphorylation targeted Ser⁷⁴⁴, whereas autophosphorylation was the predominant mechanism for Ser⁷⁴⁸ in cells stimulated with G_q-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation requires the identification of the molecular pathways that govern the transition of quiescent cells into the S phase of the cell cycle. In this context the activation and phosphorylation of protein kinase D (PKD),⁴ the founding member of a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase (CAMK) group and separate from the previously identified PKCs (for review, see Ref. 1), are attracting intense attention. In unstimulated cells, PKD is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing a repeat of cysteinerich zinc finger-like motifs and a pleckstrin homology (PH) domain (1–4). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (5–7). In response to cellular stimuli (1), including phorbol esters, growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR) agonists (6, 8–16) that signal through G_q, G₁₂, G_i, and Rho (11, 15–19), PKD is converted into a form with high catalytic activity, as shown by in vitro kinase assays performed in the absence of lipid co-activators (5, 20).During these studies multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do not directly inhibit PKD catalytic activity (5, 20), implying that PKD activation in intact cells is mediated directly or indirectly through PKCs. Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade induced by multiple GPCR agonists and other receptor ligands in a range of cell types (for review, see Ref. 1). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (1, 4, 7, 17, 21). Collectively, these findings demonstrated the existence of a rapidly activated PKC-PKD protein kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD activation was followed by a late, PKC-independent phase of catalytic activation and phosphorylation induced by stimulation of the bombesin G_q-coupled receptor ectopically expressed in COS-7 cells (22). This study raised the possibility that PKD mediates rapid biological responses downstream of PKCs, whereas, in striking contrast, PKD could mediate long term responses through PKC-independent pathways. Despite its potential importance for defining the role of PKC and PKD in signal transduction, this hypothesis has not been tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in several cellular processes and activities, including signal transduction (14, 23–25), chromatin organization (26), Golgi function (27, 28), gene expression (29–31), immune regulation (26), and cell survival, adhesion, motility, differentiation, DNA synthesis, and proliferation (for review, see Ref. 1). In Swiss 3T3 fibroblasts, a cell line used extensively as a model system to elucidate mechanisms of mitogenic signaling (32–34), PKD expression potently enhances ERK activation, DNA synthesis, and cell proliferation induced by G_q-coupled receptor agonists (8, 14). Here, we used this model system to elucidate the role and mechanism(s) of biphasic PKD activation. First, we show that the G_q-coupled receptor agonists bombesin and vasopressin, in contrast to phorbol esters, specifically induce PKD activation through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3 cells. Subsequently, we demonstrate for the first time that the PKC-independent phase of PKD activation is responsible for promoting ERK signaling and progression to DNA synthesis through an epidermal growth factor receptor (EGFR)-dependent pathway. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.     

RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA

RecF pathway proteins play an important role in the restart of stalled replication and DNA repair in prokaryotes. Following DNA damage, RecF, RecR, and RecO initiate homologous recombination (HR) by loading of the RecA recombinase on single-stranded (ss) DNA, protected by ssDNA-binding protein. The specific role of RecF in this process is not well understood. Previous studies have proposed that RecF directs the RecOR complex to boundaries of damaged DNA regions by recognizing single-stranded/double-stranded (ss/ds) DNA junctions. RecF belongs to ABC-type ATPases, which function through an ATP-dependent dimerization. Here, we demonstrate that the RecF of Deinococcus radiodurans interacts with DNA as an ATP-dependent dimer, and that the DNA binding and ATPase activity of RecF depend on both the structure of DNA substrate, and the presence of RecR. We found that RecR interacts as a tetramer with the RecF dimer. RecR increases the RecF affinity to dsDNA without stimulating ATP hydrolysis but destabilizes RecF binding to ssDNA and dimerization, likely due to increasing the ATPase rate. The DNA-dependent binding of RecR to the RecF-DNA complex occurs through specific protein-protein interactions without significant contributions from RecR-DNA interactions. Finally, RecF neither alone nor in complex with RecR preferentially binds to the ss/dsDNA junction. Our data suggest that the specificity of the RecFOR complex toward the boundaries of DNA damaged regions may result from a network of protein-protein and DNA-protein interactions, rather than a simple recognition of the ss/dsDNA junction by RecF.Homologous recombination (HR)² is one of the primary mechanisms by which cells repair dsDNA breaks (DSBs) and ssDNA gaps (SSGs), and is important for restart of stalled DNA replication (1). HR is initiated when RecA-like recombinases bind to ssDNA forming an extended nucleoprotein filament, referred to as a presynaptic complex (2). The potential for genetic rearrangements dictates that HR initiation is tightly regulated at multiple levels (1). During replication, the ssDNA-binding protein (SSB) protects transiently unwound DNA chains, preventing interactions with recombinases. Following DNA damage, recombination mediator proteins (RMPs) initiate HR by facilitating the formation of the recombinase filaments with ssDNA, while removing SSB (3, 4). Mutations in human proteins involved in HR initiation are linked to cancer predisposition, chromosome instability, UV sensitivity, and premature aging diseases (4–8). To date, little is known about the mechanism by which RMPs regulate the formation of the recombinase filaments on the SSB-protected ssDNA.In Escherichia coli, there are two major recombination pathways, RecBCD and RecF (9, 10). A helicase/nuclease RecBCD complex processes DSBs and recruits RecA on ssDNA in a sequence-specific manner (11–13). The principle players in the RecF pathway are the RecF, RecO, and RecR proteins, which form an epistatic group that is important for SSG repair, for restart of stalled DNA replication, and under specific conditions, can also process DSBs (14–20). Homologs of RecF, -O, and -R are present in the majority of known bacteria (21), including Deinococcus radiodurans, extremely radiation-resistant bacteria that lacks the RecBCD pathway, yet is capable of repairing thousands of DSBs (22, 23). In addition, the sequence or functional homologs of RecF pathway proteins are involved in similar pathways in eukaryotes that include among others WRN, BLM, RAD52, and BRCA2 proteins (4–8).The involvement of all three RecF, -O, and -R proteins in HR initiation is well documented by genetic and cellular approaches (18, 24–30), yet their biochemical functions in the initiation process remain unclear, particularly with respect to RecF. RecO and RecR proteins are sufficient to promote formation of the RecA filament on SSB-bound ssDNA in vitro (27). The UV-sensitive phenotype of recF mutants can be suppressed by RecOR overexpression, suggesting that RecF may direct the RMP complex to DNA-damaged regions where HR initiation is required (31). In agreement with this hypothesis, RecF dramatically increases the efficiency of the RecA loading at ds/ssDNA junctions with a 3′ ssDNA extension under specific conditions (32). RecF and RecR proteins also prevent the RecA filaments from extending into dsDNA regions adjacent to SSGs (33). These data suggest that RecF may directly recognize an ss/dsDNA junction structure (34). However, DNA binding experiments have not provided clear evidence to support such a hypothesis (11).The targeting promoted by RecF may also occur through more complex processes. RecF shares a high structural similarity with the head domain of Rad50, an ABC-type ATPase that recognizes DSBs and initiates repair in archaea and eukaryotes (35). All known ABC-type ATPases function as oligomeric complexes in which a sequence of inter- and intra-molecular interactions is triggered by the ATP-dependent dimerization and the dimer-dependent ATP hydrolysis (36–39). RecF is also an ATP-dependent DNA-binding protein and a weak DNA-dependent ATPase (11, 40). RecF forms an ATP-dependent dimer and all three conserved motifs (Walker A, Walker B, and “signature”) of RecF are important for ATP-dependent dimerization, ATP hydrolysis, and functional resistance to DNA damage (35). Thus, RecF may function in recombination initiation through a complex pathway of protein-protein and DNA-protein interactions regulated by ATP-dependent RecF dimerization.In this report, we present a detailed characterization of the RecF dimerization, and its role in the RecF interaction with various DNA substrates, with RecR, and in ATP hydrolysis. Our data outline the following key findings. First, RecF interacts with DNA as a dimer. Second, neither RecF alone nor the RecFR complex preferentially binds the ss/dsDNA junction. Finally, RecR changes the ATPase activity and the DNA binding of RecF by destabilizing the interaction with ssDNA, and greatly enhancing the interaction with dsDNA. Our results suggest that the specificity of RecF for the boundaries of SSGs is likely to result from a sequence of protein-protein interaction events rather than a simple RecF ss/dsDNA binding, underlining a highly regulated mechanism of the HR initiation by the RecFOR proteins.     

Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL

The RAD51 protein is a central player in homologous recombinational repair. The RAD51B protein is one of five RAD51 paralogs that function in the homologous recombinational repair pathway in higher eukaryotes. In the present study, we found that the human EVL (Ena/Vasp-like) protein, which is suggested to be involved in actin-remodeling processes, unexpectedly binds to the RAD51 and RAD51B proteins and stimulates the RAD51-mediated homologous pairing and strand exchange. The EVL knockdown cells impaired RAD51 assembly onto damaged DNA after ionizing radiation or mitomycin C treatment. The EVL protein alone promotes single-stranded DNA annealing, and the recombination activities of the EVL protein are further enhanced by the RAD51B protein. The expression of the EVL protein is not ubiquitous, but it is significantly expressed in breast cancer-derived MCF7 cells. These results suggest that the EVL protein is a novel recombination factor that may be required for repairing specific DNA lesions, and that may cause tumor malignancy by its inappropriate expression.Chromosomal DNA double strand breaks (DSBs)² are potential inducers of chromosomal aberrations and tumorigenesis, and they are accurately repaired by the homologous recombinational repair (HRR) pathway, without base substitutions, deletions, and insertions (1–3). In the HRR pathway (4, 5), single-stranded DNA (ssDNA) tails are produced at the DSB sites. The RAD51 protein, a eukaryotic homologue of the bacterial RecA protein, binds to the ssDNA tail and forms a helical nucleoprotein filament. The RAD51-ssDNA filament then binds to the intact double-stranded DNA (dsDNA) to form a three-component complex, containing ssDNA, dsDNA, and the RAD51 protein. In this three-component complex, the RAD51 protein promotes recombination reactions, such as homologous pairing and strand exchange (6–9).The RAD51 protein requires auxiliary proteins to promote the homologous pairing and strand exchange reactions efficiently in cells (10–12). In humans, the RAD52, RAD54, and RAD54B proteins directly interact with the RAD51 protein (13–17) and stimulate the RAD51-mediated homologous pairing and/or strand exchange reactions in vitro (18–21). The human RAD51AP1 protein, which directly binds to the RAD51 protein (22), was also found to stimulate RAD51-mediated homologous pairing in vitro (23, 24). The BRCA2 protein contains ssDNA-binding, dsDNA-binding, and RAD51-binding motifs (25–33), and the Ustilago maydis BRCA2 ortholog, Brh2, reportedly stimulated RAD51-mediated strand exchange (34, 35). Most of these RAD51-interacting factors are known to be required for efficient RAD51 assembly onto DSB sites in cells treated with ionizing radiation (10–12).The RAD51B (RAD51L1, Rec2) protein is a member of the RAD51 paralogs, which share about 20–30% amino acid sequence similarity with the RAD51 protein (36–38). RAD51B-deficient cells are hypersensitive to DSB-inducing agents, such as cisplatin, mitomycin C (MMC), and γ-rays, indicating that the RAD51B protein is involved in the HRR pathway (39–44). Genetic experiments revealed that RAD51B-deficient cells exhibited impaired RAD51 assembly onto DSB sites (39, 44), suggesting that the RAD51B protein functions in the early stage of the HRR pathway. Biochemical experiments also suggested that the RAD51B protein participates in the early to late stages of the HRR pathway (45–47).In the present study, we found that the human EVL (Ena/Vasp-like) protein binds to the RAD51 and RAD51B proteins in a HeLa cell extract. The EVL protein is known to be involved in cytoplasmic actin remodeling (48) and is also overexpressed in breast cancer (49). Like the RAD51B knockdown cells, the EVL knockdown cells partially impaired RAD51 foci formation after DSB induction, suggesting that the EVL protein enhances RAD51 assembly onto DSB sites. The purified EVL protein preferentially bound to ssDNA and stimulated RAD51-mediated homologous pairing and strand exchange. The EVL protein also promoted the annealing of complementary strands. These recombination reactions that were stimulated or promoted by the EVL protein were further enhanced by the RAD51B protein. These results strongly suggested that the EVL protein is a novel factor that activates RAD51-mediated recombination reactions, probably with the RAD51B protein. We anticipate that, in addition to its involvement in cytoplasmic actin dynamics, the EVL protein may be required in homologous recombination for repairing specific DNA lesions, and it may cause tumor malignancy by inappropriate recombination enhanced by EVL overexpression in certain types of tumor cells.     

Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.Matrix metalloproteinases (MMP)² are extracellular hydrolytic enzymes involved in a variety of processes including connective tissue cleavage and remodeling (1–3). All 23 members of the family are able to cleave simple peptides derived from connective tissue components such as collagen, gelatin, elastin, etc. A subset of MMPs is able to hydrolyze more resistant polymeric substrates, such as cross-linked elastin, and partially degraded collagen forms, such as gelatin and type IV collagens (4). Intact triple helical type I–III collagen is only attacked by collagenases MMP-1, MMP-8, and MMP-13 and by MMP-2 and MMP-14 (5–12). Although the detailed mechanism of cleavage of single chain peptides by MMP has been largely elucidated (13–19), little is known about the process of hydrolysis of triple helical collagen. In fact, triple helical collagen cannot be accommodated in the substrate-binding groove of the catalytic site of MMPs (9).All MMPs (but MMP-7) in their active form are constituted by a catalytic domain (CAT) and a hemopexin-like domain (HPX) (20–22). The CAT domain contains two zinc ions and one to three calcium ions. One zinc ion is at the catalytic site and is responsible for the activity, whereas the other metal ions have structural roles. The isolated CAT domains retain full catalytic activity toward simple peptides and single chain polymeric substrates such as elastin, whereas hydrolysis of triple helical collagen also requires the presence of the HPX domain (9, 23–25). It has been shown that the isolated CAT domain regains a small fraction of the activity of the full-length (FL) protein when high amounts of either inactivated full-length proteins or isolated HPX domains are added to the assay solution (9). Finally, it has been shown that the presence of the HPX domain alone alters the CD spectrum of triple helical collagen in a way that suggests its partial unwinding (26, 27). It is tempting to speculate that full-length collagenases attack collagen by first locally unwinding the triple helical structure with the help of the HPX domain and then cleaving the resulting, exposed, single filaments (9, 28).Until 2007, three-dimensional structures of full-length MMPs had been reported only for collagenase MMP-1 (29–31) and gelatinase MMP-2 (32). The structures of the two proteins are very similar and show a compact arrangement of the two domains, which are connected by a short linker (14 and 20 amino acids, respectively). It is difficult to envisage that rigid and compact molecules of this type can interact with triple helical collagen in a way that can lead to first unwinding and then cleavage of individual filaments. It has been recently suggested that such concerted action could occur much more easily if the two domains could enjoy at least a partial conformational independence (9). Slight differences in the reciprocal orientation of the CAT and HPX domains of MMP-1 in the presence (29) and absence (30, 31) of the prodomain were indeed taken as a hint that the two domains could experience relative mobility (29).Two recent solution studies have shown that conformational independence is indeed occurring in gelatinase MMP-9 (33) and elastase MMP-12 (34), whereas the x-ray structure of the latter (34) is only slightly less compact than those of MMP-1 (29–31) and MMP-2 (32). Among MMPs, MMP-9 features an exceptionally long linker (68 amino acid) (33, 35), which in fact constitutes a small domain by itself (the O-glycosylated domain) (33), and therefore, this inspiring observation can hardly be taken as evidence that conformational freedom is a general characteristic of the two-domain MMPs. MMP-12 features a much more normal 16-amino acid linker, thereby making more probable a general functional role for this conformational freedom (34). However, both MMP-9 and MMP-12 retain their full catalytic activity against their substrates even when deprived of the HPX domain (9). Therefore, the question remains of whether conformational freedom is also a required characteristic for those MMPs that are only active as full-length proteins, i.e. collagenases. Interestingly, the three collagenases (MMP-1, MMP-8, and MMP-13) have the shortest linker (14 amino acids) among all MMPs. Demonstrating or negating the presence of conformational freedom in one of these collagenases would therefore constitute a significant step forward to formulate mechanistic hypotheses on their collagenolytic activity.Our recent studies on MMP-12 in solution (34) have shown that a combination of NMR relaxation studies and small angle x-ray scattering (SAXS) is enough to show the presence and the extent of the relative conformational freedom of the two domains of MMPs. Here we apply the same strategy to full-length MMP-1 and show that sizable conformational freedom is indeed experienced even by this prototypical collagenase, although somewhat less pronounced than that observed for MMP-12.     

The Structure of an Interdomain Complex That Regulates Talin Activity

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in regulating the affinity of the integrin family of cell adhesion molecules for ligand (1) and in coupling integrins to the actin cytoskeleton (2). Thus, depletion of talin results in defects in integrin activation (3), integrin signaling through focal adhesion kinase, the maintenance of cell spreading, and the assembly of focal adhesions in cultured cells (4). In the whole organism, studies on the single talin gene in worms (5) and flies (6) show that talin is essential for a variety of integrin-mediated events that are crucial for normal embryonic development. In vertebrates, there are two talin genes, and mice carrying a talin1 null allele fail to complete gastrulation (7). Tissue-specific inactivation of talin1 results in an inability to activate integrins in platelets (8, 9), defects in the membrane-cytoskeletal interface in megakaryocytes (10), and disruption of the myotendinous junction in skeletal muscle (11). In contrast, mice homozygous for a talin2 gene trap allele have no phenotype, although the allele may be hypomorphic (12).Recent structural studies have provided substantial insights into the molecular basis of talin action. Talin is composed of an N-terminal globular head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The talin head contains a FERM² domain (made up of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0 (2). Studies by Wegener et al. (30) have shown how the F3 FERM subdomain, which has a phosphotyrosine binding domain fold, interacts with both the canonical NPXY motif and the membrane-proximal helical region of the cytoplasmic tails of integrin β-subunits (13). The latter interaction apparently activates the integrin by disrupting the salt bridge between the integrin α- and β-subunit tails that normally keeps integrins locked in a low affinity state. The observation that the F0 region is also important in integrin activation (14) may be explained by our recent finding that F0 binds, albeit with low affinity, Rap1-GTP,³ a known activator of integrins (15, 16). The talin rod is made up of a series of amphipathic α-helical bundles (17–20) and contains a second integrin binding site (IBS2) (21), numerous binding sites for the cytoskeletal protein vinculin (22), at least two actin binding sites (23), and a C-terminal helix that is required for assembly of talin dimers (20, 24).Both biochemical (25) and cellular studies (16) suggest that the integrin binding sites in full-length talin are masked, and both phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in exposing these sites. It is well established that some members of the FERM domain family of proteins are regulated by a head-tail interaction (26); gel filtration, sedimentation velocity, and electron microscopy studies all show that talin is globular in low salt buffers, although it is more elongated (∼60 nm in length) in high salt (27). By contrast, the talin rod liberated from full-length talin by calpain-II cleavage is elongated in both buffers, indicating that the head is required for talin to adopt a more compact state. Direct evidence for an interaction between the talin head and rod has recently emerged from NMR studies by Goksoy et al. (28), who demonstrated binding of ¹⁵N-labeled talin F3 to a talin rod fragment spanning residues 1654–2344, an interaction that was confirmed by surface plasmon resonance (K_d = 0.57 μm) (28). Chemical shift data also showed that this segment of the talin rod partially masked the binding site in F3 for the membraneproximal helix of the β3-integrin tail (28), directly implicating the talin head-rod interaction in regulating the integrin binding activity of talin. Goksoy et al. (28) subdivided the F3 binding site in this rod fragment into two sites with higher affinity (K_d ∼3.6 μm; residues 1654–1848) and lower affinity (K_d ∼78 μm; residues 1984–2344). Here, we define the rod domain boundaries and determine the NMR structure of residues 1655–1822, a five-helix bundle. We further show that this domain binds F3 predominantly via surface-exposed residues on helix 4, with an affinity similar to the high affinity site reported by Goksoy et al. (28). We also report the structure of the complex between F3 and the rod domain and show that the latter masks the known binding site in F3 for the β3-integrin tail and is expected to inhibit the association of the talin FERM domain with the membrane.