Conserved Threonine Residue Research Articles

Kinetic and Structural Insights into the Mechanism of AMPylation by VopS Fic Domain

The bacterial pathogen Vibrio parahemeolyticus manipulates host signaling pathways during infections by injecting type III effectors into the cytoplasm of the target cell. One of these effectors, VopS, blocks actin assembly by AMPylation of a conserved threonine residue in the switch 1 region of Rho GTPases. The modified GTPases are no longer able to interact with downstream effectors due to steric hindrance by the covalently linked AMP moiety. Herein we analyze the structure of VopS and its evolutionarily conserved catalytic residues. Steady-state analysis of VopS mutants provides kinetic understanding on the functional role of each residue for AMPylation activity by the Fic domain. Further mechanistic analysis of VopS with its two substrates, ATP and Cdc42, demonstrates that VopS utilizes a sequential mechanism to AMPylate Rho GTPases. Discovery of a ternary reaction mechanism along with structural insight provides critical groundwork for future studies for the family of AMPylators that modify hydroxyl-containing residues with AMP.

Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase,

Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin.

Vasopressin‐mediated signaling in the distal convoluted tubule regulates phosphorylation of the thiazide‐sensitive Na+‐Cl− cotransporter (NCC) at two conserved threonine residues

The Na+‐Cl− cotransporter (NCC) is important for renal electrolyte balance. Phosphorylation of NCC has been proposed to play a major role in its transport activity and cellular localization. Using recently generated phospho‐specific antibodies against two conserved N‐terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) we assessed the role of vasopressin (AVP) in regulating NCC in vivo. In AVP‐deficient Brattleboro rats, acute dDAVP exposure for 15 min significantly increased pNCC abundance at the apical plasma membrane approximately 300%. Acute dDAVP did not alter the abundance of the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) previously implicated in NCC phosphorylation. Early and late DCTs increased their intracellular Ca2+ levels in response to 1 min of superfusion of dDAVP (2 × 10−10 M), confirming that the DCT is an AVP responsive segment. In rats fed a high salt diet alongside AT1‐R blockade by candesartan, similar increases in pNCC abundance were observed following chronic or acute dDAVP. Candesartan treatment reduced the abundances of total SPAK and pSPAK, suggesting that this kinase can be regulated via the AT1‐R in vivo. Taken together our results indicate that AVP is a potent regulator of NCC activity.Funding is provided by the Danish Medical Research Council, the Novo Nordisk Fond and the Carlsberg Foundation.

A Conserved Threonine Residue in the Juxtamembrane Domain of the XA21 Pattern Recognition Receptor Is Critical for Kinase Autophosphorylation and XA21-mediated Immunity

Despite the key role that pattern recognition receptors (PRRs) play in regulating immunity in plants and animals, the mechanism of activation of the associated non-arginine-aspartate (non-RD) kinases is unknown. The rice PRR XA21 recognizes the pathogen-associated molecular pattern, Ax21 (activator of XA21-mediated immunity). Here we show that the XA21 juxtamembrane (JM) domain is required for kinase autophosphorylation. Threonine 705 in the XA21 JM domain is essential for XA21 autophosphorylation in vitro and XA21-mediated innate immunity in vivo. The replacement of Thr(705) by an alanine or glutamic acid abolishes XA21 autophosphorylation and eliminates interactions between XA21 and four XA21-binding proteins in yeast and rice. Although threonine residues analogous to Thr(705) of XA21 are present in the JM domains of most RD and non-RD plant receptor-like kinases, this residue is not required for autophosphorylation of the Arabidopsis RD RLK BRI1 (brassinosteroid insensitive 1). The threonine 705 of XA21 is conserved only in the JM domains of plant RLKs but not in those of fly, human, or mouse suggesting distinct regulatory mechanisms. These results contribute to growing knowledge regarding the mechanism by which non-RD RLKs function in plant.

The N Terminus of Monoamine Transporters Is a Lever Required for the Action of Amphetamines

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.

Schistosoma mansoni Polo-like kinase 1: A mitotic kinase with key functions in parasite reproduction

Polo-like kinases (Plks) are conserved regulators of mitosis. In mammals, Plk1 is over-expressed in a wide range of tumour cells and constitutes a valuable target for anti-cancer therapy. This work presents the characterisation of the Plk1 homologue (SmPlk1) of Schistosoma mansoni, a trematode responsible for schistosomiasis, one of the most important parasitic diseases, second only to malaria. The intense levels of disease transmission and the severity of pathologies are the consequences of the exceptional reproductive activity of schistosomes, in which Plks may play a decisive role. Structural and functional analyses of SmPlk1 have demonstrated its homology with other Plk1 members and its conserved function in mitotic processes. Activation of SmPlk1 was shown to be dependent on phosphorylation of its conserved threonine residue (T 182) and the ability of active SmPlk1 to induce mitosis was demonstrated in the Xenopus oocyte model. SmPlk1 transcripts were detected abundantly in parasite stages containing a high amount of germinal cells. A potential role of SmPlk1 in mitosis and/or meiosis in schistosomes was supported by the in situ detection of SmPlk1 transcripts in female vitelline cells and oocytes as well as in male spermatocytes. Several Plk inhibitors were shown to inhibit SmPlk1 activity in Xenopus oocytes, and BI 2536 (the first-in-class prototype Plk1 inhibitor) induced in vitro dramatic alterations in schistosome gonads, which affected oogenesis and spermatogenesis. These results indicate a major role for SmPlk1 in parasite reproduction and suggest its importance as a potential new target against schistosomiasis.

PP1 phosphatase-binding motif in Reg1 protein of Saccharomyces cerevisiae is required for interaction with both the PP1 phosphatase Glc7 and the Snf1 protein kinase

In Saccharomyces cerevisiae, Snf1 kinase, the ortholog of the mammalian AMP-activated protein kinase, is activated by an increase in the phosphorylation of the conserved threonine residue in its activation loop. The phosphorylation status of this key site is determined by changes in the rate of dephosphorylation catalyzed by the yeast PP1 phosphatase Glc7 in a complex with the Reg1 protein. Reg1 and many PP1 phosphatase regulatory subunits utilize some variation of the conserved RVxF motif for interaction with PP1. In the Snf1 pathway, the exact role of the Reg1 protein is uncertain since it binds to both the Glc7 phosphatase and to Snf1, the Glc7 substrate. In this study we sought to clarify the role of Reg1 by separating the Snf1- and Glc7-binding functions. We generated a series of Reg1 proteins, some with deletions of conserved domains and one with two amino acid changes in the RVxF motif. The ability of Reg1 to bind Snf1 and Glc7 required the same domains of Reg1. Further, the RVxF motif that is essential for Reg1 binding to Glc7 is also required for binding to Snf1. Our data suggest that the regulation of Snf1 dephosphorylation is imparted through a dynamic competition between the Glc7 phosphatase and the Snf1 kinase for binding to the PP1 regulatory subunit Reg1.

Identification of substrates for cyclin dependent kinases

Protein phosphorylation is a key regulatory mechanism for cell cycle control in eukaryotes. From yeast to humans, cell cycle progression and cell division require the activation of a group of serine– threonine protein kinases called cyclin dependent kinases (CDKs) (Morgan, 1997), which initiate and coordinate these processes by orderly phosphorylation of their targets. CDKs require association with a cyclin subunit to activate them,whichprovides substrate specificity (Russo,1997; Solomonet al.,1992), and phosphorylation by an activating protein kinase (CAK) at a conserved threonine residue (Krek and Nigg,1992; Lorca et al., 1992; Solomon et al., 1992). The concentration of cyclins oscillate during the cell cycle and their abundance is regulated at several levels: transcriptional, translational and at the level of protein stability. In particular, at the end of mitosis, ubiquitin-mediated proteolysis results in the disappearance of mitotic cyclins, which results in inactivation of CDKs (Zachariae and Nasmyth, 1999). The key cell cycle transitions involving the activity of CDKs occur at the initiation of S-phase and entry into mitosis. Loss of CDK activity at the end of mitosis permits the return to interphase, and is presumably accompanied by dephosphorylation of the majority of target proteins that were modified when the cells entered mitosis. There is rather specific control of the different phases of the cell cycle, so that cells do not enter mitosis with unreplicated or damaged DNA, and do not exit mitosis until all the chromosomes are correctly aligned on the metaphase plate. There is a theoretical conundrum, however, in explaining how it is that activation of S-phase CDKs, such as cyclin E/CDK2 and cyclin A/ CDK2 in vertebrate cells promotes entry into S-phase and not into mitosis, or why activation of the Mphase CDKs does not promote a further round of DNA replication. One hypothesis to explain why cells enter S-phase, rather than mitosis, when the S-phase CDK (CDK2-cyclin E in higher eukaryotes) is activated would be a restricted substrate specificity for this particular CDK, yet there is very little information on this point. Another possibility is that CDK2-cyclin E resides in the nucleus, and entry

The glutamate switch is present in all seven clades of AAA+ protein.

Recent work has identified a "glutamate switch" in six of the seven clades of AAA+ ATPases. The glutamate switch acts to transduce information regarding substrate binding to the ATPase active site. We provide biochemical evidence that a highly conserved threonine residue acts as a glutamate switch in the replicative helicase, MCM, and, thus, reveal that the glutamate switch is a feature common to all seven AAA+ clades.

Conserved Threonine Residues within the A-Loop of the Receptor NIK Differentially Regulate the Kinase Function Required for Antiviral Signaling

NSP-interacting kinase (NIK1) is a receptor-like kinase identified as a virulence target of the begomovirus nuclear shuttle protein (NSP). We found that NIK1 undergoes a stepwise pattern of phosphorylation within its activation-loop domain (A-loop) with distinct roles for different threonine residues. Mutations at Thr-474 or Thr-468 impaired autophosphorylation and were defective for kinase activation. In contrast, a mutation at Thr-469 did not impact autophosphorylation and increased substrate phosphorylation, suggesting an inhibitory role for Thr-469 in kinase function. To dissect the functional significance of these results, we used NSP-expressing virus infection as a mechanism to interfere with wild type and mutant NIK1 action in plants. The NIK1 knockout mutant shows enhanced susceptibility to virus infections, a phenotype that could be complemented with ectopic expression of a 35S-NIK1 or 35S-T469A NIK1 transgenes. However, ectopic expression of an inactive kinase or the 35S-T474A NIK1 mutant did not reverse the enhanced susceptibility phenotype of knockout lines, demonstrating that Thr-474 autophosphorylation was needed to transduce a defense response to geminiviruses. Furthermore, mutations at Thr-474 and Thr-469 residues antagonistically affected NIK-mediated nuclear relocation of the downstream effector rpL10. These results establish that NIK1 functions as an authentic defense receptor as it requires activation to elicit a defense response. Our data also suggest a model whereby phosphorylation-dependent activation of a plant receptor-like kinase enables the A-loop to control differentially auto- and substrate phosphorylation.

Autoinhibition and Autoactivation of the DNA Replication Checkpoint Kinase Cds1

Cds1 is the ortholog of Chk2 and the major effector of the DNA replication checkpoint in Schizosaccharomyces pombe. Previous studies have shown that Cds1 is activated by a two-stage mechanism. In the priming stage, the sensor kinase Rad3 and the mediator Mrc1 function to phosphorylate a threonine residue, Thr(11), in the SQ/TQ domain of Cds1. In the autoactivation stage, primed Cds1 molecules dimerize via intermolecular interactions between the phosphorylated Thr(11) in one Cds1 and the forkhead-associated domain of the other. Dimerization activates Cds1, probably by promoting autophosphorylation. To define the mechanisms for the autoactivation of primed Cds1 and the regulation of this process, we carried out genetic and biochemical studies to identify phosphorylatable residues required for checkpoint activation. Our data indicate that dimerization of Cds1 promotes trans-autophosphorylation of a number of residues in the catalytic domain, but phosphorylation of a highly conserved threonine residue (Thr(328)) in the activation loop is the only covalent modification required for kinase activation in vitro and in vivo. Autophosphorylation of Thr(328) and kinase activation in unprimed, monomeric Cds1 are strongly inhibited by the C-terminal 27-amino acid tail of the enzyme. This autoinhibitory effect may play an important role in preventing spontaneous activation of the replication checkpoint during normal cell cycles. The two-stage activation pathway and the autoinhibition mechanism, which are probably shared by other members of the Chk2 family, provide sensitivity, specificity, and noise immunity, properties required for the replication checkpoint.

Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic "arginine finger" seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099-1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42.GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.

Mechanism of Activation and Functional Role of Protein Kinase Cη in Human Platelets

The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

The regulation and function of mammalian AMPK‐related kinases

AMP-activated protein kinase (AMPK) is a key regulator of cellular and whole-body energy homeostasis. Recently, 12 AMPK-related kinases (BRSK1, BRSK2, NUAK1, NUAK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) were identified that are closely related by sequence homology to the catalytic domain of AMPK. The protein kinase LKB1 acts as a master upstream kinase activating AMPK and 11 of the AMPK-related kinases by phosphorylation of a conserved threonine residue in their T-loop region. Further sequence analyses have identified the eight-member SNRK kinase family as distant relatives of AMPK. However, only one of these is phosphorylated and activated by LKB1. Although much is known about AMPK, many of the AMPK-related kinases remain largely uncharacterized. This review outlines the general similarities in structure and function of the AMPK-related kinases before examining the specific characteristics of each, including a brief discussion of the SNRK family.

The Mycobacterium tuberculosis Protein Kinase K Modulates Activation of Transcription from the Promoter of Mycobacterial Monooxygenase Operon through Phosphorylation of the Transcriptional Regulator VirS

Mycobacterium tuberculosis encodes for 11 eukaryotic-like serine/threonine protein kinases. Genetic and biochemical studies show that these kinases regulate various cellular processes including cell shape and morphology, glucose and glutamine transport, phagosome-lysosome fusion and the expression, and/or activity of transcription factors. PknK is the largest predicted serine/threonine protein kinase in M. tuberculosis. Here, we have cloned, overexpressed, and purified protein kinase K (PknK) to near homogeneity and show that its ability to phosphorylate proteins is dependent on the invariant lysine (Lys(55)), and on two conserved threonine residues present in its activation loop. Despite being devoid of any apparent transmembrane domain, PknK is localized to the cell wall fraction, suggesting probable anchoring of the kinase to the cell membrane region. The pknK gene is located in the vicinity of the virS gene, which is known to regulate the expression of the mycobacterial monooxygenase (mymA) operon. We report here for the first time that VirS is in fact a substrate of PknK. In addition, four of the proteins encoded by mymA operon are also found to be substrates of PknK. Results show that VirS is a bona fide substrate of PknK in vivo, and PknK-mediated phosphorylation of VirS increases its affinity for mym promoter DNA. Reporter assays reveal that PknK modulates VirS-mediated stimulation of transcription from the mym promoter. These findings suggest that the expression of mymA operon genes is regulated through PknK-mediated phosphorylation of VirS.

Arabidopsis Protein Kinases GRIK1 and GRIK2 Specifically Activate SnRK1 by Phosphorylating Its Activation Loop

SNF1-related kinases (SnRK1s) play central roles in coordinating energy balance and nutrient metabolism in plants. SNF1 and AMPK, the SnRK1 homologs in budding yeast (Saccharomyces cerevisiae) and mammals, are activated by phosphorylation of conserved threonine residues in their activation loops. Arabidopsis (Arabidopsis thaliana) GRIK1 and GRIK2, which were first characterized as geminivirus Rep interacting kinases, are phylogenetically related to SNF1 and AMPK activating kinases. In this study, we used recombinant proteins produced in bacteria to show that both GRIKs specifically bind to the SnRK1 catalytic subunit and phosphorylate the equivalent threonine residue in its activation loop in vitro. GRIK-mediated phosphorylation increased SnRK1 kinase activity in autophosphorylation and peptide substrate assays. These data, together with earlier observations that GRIKs could complement yeast mutants lacking SNF1 activation activities, established that the GRIKs are SnRK1 activating kinases. Given that the GRIK proteins only accumulate in young tissues and geminivirus-infected mature leaves, the GRIK-SnRK1 cascade may function in a developmentally regulated fashion and coordinate the unique metabolic requirements of rapidly growing cells and geminivirus-infected cells that have been induced to reenter the cell cycle.

Hunting for the SNARK in metabolic disease

dysregulation of energy balance is a primary constituent in the etiology of obesity and type 2 diabetes mellitus, which is manifested by altered metabolic homeostasis and insulin resistance in a variety of tissues, including brain, liver, and skeletal muscle. The discovery of the AMP-activated

AMPylation of Rho GTPases by Vibrio VopS Disrupts Effector Binding and Downstream Signaling

The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine 5'-monophosphate (AMP). The resulting AMPylation prevented the interaction of Rho GTPases with downstream effectors, thereby inhibiting actin assembly in the infected cell. Eukaryotic proteins were also directly modified with AMP, potentially expanding the repertoire of posttranslational modifications for molecular signaling.

Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association.

The Par-1 protein kinases are conserved from yeast to humans, where they function as key polarity determinants. The mammalian Par-1 family is comprised of 4 members (Par-1a, -b, -c, and -d). Previously, we demonstrated that atypical protein kinase C (aPKC) phosphorylates the Par-1 kinases on a conserved threonine residue (T595) to regulate localization and kinase activity. Here, we demonstrate that Par-1b is also regulated by another arm of the PKC pathway, one that involves novel PKCs (nPKC) and protein kinase D. Treatment of cells with the PKC activator phorbol-12-myristate-13-acetate (PMA) potently stimulated phosphorylation of Par-1b on serine 400 (S400), a residue that is conserved in all 4 mammalian Par-1 kinases as well as the fly ortholog. We demonstrate that PMA stimulates nPKC to activate PKD, which in turn directly phosphorylates Par-1b on S400 to positively regulate 14-3-3 binding and to negatively regulate membrane association. Thus, 2 arms of the PKC pathway regulate interactions between Par-1b and 14-3-3 proteins: one involving aPKC and the other nPKC/PKD.

Identification of an Evolutionarily Conserved Extracellular Threonine Residue Critical for Surface Expression and Its Potential Coupling of Adjacent Voltage-sensing and Gating Domains in Voltage-gated Potassium Channels

The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.