Browsing by Subject "Protein Kinases"
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Item AXL Targeting Restores PD-1 Blockade Sensitivity of STK11/LKB1 Mutant NSCLC Through Expansion of TCF1+ CD8 T Cells(December 2021) Li, Huiyu; Akbay, Esra A.; DeBerardinis, Ralph J.; Fu, Yang-Xin; Brekken, Rolf A.; Minna, John D.; Aguilera, Todd A.Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB) for unknown reasons. We found that introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically, this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. We found that systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expands tumor-associated TCF1+ PD-1+ CD8 T cells, restoring therapeutic response to PD-1 ICB for KPL tumors. This effect was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. Anecdotal NSCLC patients with STK11/LKB1 mutant tumors also demonstrated responses to the combination of AXL inhibitor bemcentinib and pembrolizumab. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.Item Biophysical and Biochemical Characterization of a REC Domain: Unfolded to Folded Transition of EL_LovR(2014-08-18) Ocasio, Victor J.; Hendrixson, David R.; Gardner, Kevin H.; Sperandio, Vanessa; Rizo-Rey, JoséProkaryotes frequently use two component systems to couple environmental stimuli to adaptive responses. These pathways use histidine kinases to detect environmental cues, harnessing these to control phosphorylation of the receiver domain of the response regulator, which convert this signal into a physiological response. Knowledge of how phosphorylation shifts receiver domains between their inactive and active states is limited, chiefly assembled from several prototypical receiver domains that switch between two similar and well-folded structures. However, it remains unclear how general these observations apply to other receiver domains, particularly for full-length proteins. Here we present a blue light-regulated two-component system from the marine α-proteobacterium Erythrobacter litoralis HTCC2594. The sensor domain of the 3 histidine kinases found in E. litoralis contain a LOV (Light-Oxygen-Voltage) domain, part of the widely used PAS (Per-ARNT-Sim) family of environmental sensors. Interestingly, one of the histidine kinases (EL362) contains a naturally occurring glycine to arginine mutation in the LOV domain that prevents chromophore binding, resulting in a "blind" histidine kinase. Reverting the arginine to a glycine residue allows blue light to trigger the autophosphorylation of EL362 and subsequent phosphotransfer towards the cognate response regulator EL_LovR. This arrangement of RRs is reminiscent of similar systems used in other bacterial general stress responses, most of which have been characterized entirely with genetic methods. Notably, EL_LovR is a single domain response regulator proposed to play a critical role in shutting off such systems via a potent phosphatase activity. Size exclusion chromatography, light scattering and NMR experiments show that phosphorylation and Mg(II) transitions EL_LovR between unfolded and folded monomeric states. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink. Taken together, our findings provide evidence that EL_LovR undergoes drastic conformational changes that have not been seen in other response regulators, likely with effects on its autophosphatase activity. In conclusion, our work expands the kinds of conformational changes and regulation used by receiver domains, critical components of bacterial signaling systems.Item Casein Kinase I Transduces WNT Signals(2005-08-11) Peters, John Michael; Graff, Jonathan M.Wnt signaling controls a diverse array of processes including cell growth, oncogenesis, and development. Components of the Wnt cascade are altered in several human cancers including colon cancers and melanomas. We set out to identify novel components of this signal transduction pathway via expression cloning in Xenopus embryos. This assay identified a protein kinase, casein kinase I (CKI), as a novel component of the Wnt signaling pathway. We first showed that expression of CKI produces Wnt phenotypes. First, CKI induced completely formed second axes, or Siamese twins, when injected into embryos. Second, embryos which had been ventralized by UV-irradiation were completely rescued by CKI, producing normal embryos. In order to extend these observations, we tested CKI in three biochemical assays of Wnt signaling. First, CKI expression stabilized b-catenin protein levels, a hallmark of Wnt signaling. Second, CKI induced the expression of Siamois and Xnr3, direct transcriptional targets of Wnt signaling. Finally, CKI expression dorsalized the ventral marginal zone in Xenopus, inducing markers of the Spemann organizer which is a product of Wnt signaling. We next investigated the ability of CKI inhibitors to block Wnt signaling in vivo. Two dominant negative forms of CKI and a specific pharmacological inhibitor of CKI all blocked the ability of Xwnt8 to induce dorsal axes and target genes in embryos. These data demonstrate that CKI function is required for transduction of Wnt signals. Additionally, we also demonstrated that CKI function is required for Wnt signaling in the nematode C. elegans, proving that CKI function in this pathway is conserved from invertebrates to vertebrates. We then investigated the biochemical mechanism of CKI function. Epistasis experiments indicated that CKI acts between Dishevelled and GSK-3. Yeast twohybrid assays showed that CKI strongly binds Dishevelled. Through in vivo phosphorylation experiments, we showed that CKI increases Disheveled phosphorylation and in vitro experiments showed that CKI can directly phosphorylate Dishevelled. To extend our studies we performed further experiments using additional CKI isoforms and found that many other isoforms can also reproduce markers of Wnt signaling. In summary, through a combination of gain-of-function and loss-of-function studies in invertebrates and vertebrates, we have shown that CKI is a novel and conserved component of the Wnt signaling pathway.Item Evaluation and Characterization of Novel Signal Transduction Pathways in Striatum(2008-05-13) Sahin, Bogachan; Bibb, James A.In the mammalian central nervous system, protein kinases and protein phosphatases control the function of myriad target proteins in the pre- and postsynaptic compartments, including other protein kinases and phosphatases, neurotransmitter receptors, ion channels, transporters, metabolic enzymes, transcription factors, cytoskeletal elements, and vesicle-docking proteins. Using biochemical and pharmacological approaches, a number of novel striatal signal transduction pathways were evaluated and characterized in the following studies, with emphasis on protein kinase C-mediated signaling. 1) A known and novel form of mouse Adk encoding splice variants of adenosine kinase, the principal enzyme of adenosine metabolism, were cloned from a mouse brain cDNA library and expressed and purified as recombinant proteins with high enzymatic activity. The tissue distribution of adenosine kinase isoform expression was defined. A polyclonal anti adenosine kinase antibody was generated for further characterization of the enzyme. In vitro protein phosphorylation studies using purified protein kinases and in vivo radioimmunoprecipitation assays using the novel antibody for adenosine kinase indicated, however, that this metabolic enzyme is unlikely to be regulated by phosphorylation. 2) Further studies using a candidate approach demonstrated the regulation of several postsynaptic phosphoproteins by striatal adenosine A2A receptor signaling, including ionotropic glutamate receptor subunits, mitogen-activated protein kinase isoforms, a striatal inhibitor of protein phosphatase 1, a protein phosphatase 1- and actin-binding protein, and the cAMP-response element-binding protein. 3) In parallel studies, inhibitor-1, a protein phosphatase 1 inhibitor activated by cAMPdependent protein kinase, was characterized as a novel protein kinase C substrate in vitro and in vivo. Phosphorylation state-specific antibodies raised against this novel phosphorylation site showed that it is dephosphorylated by protein phosphatase 1 and positively regulated by group I metabotropic glutamate receptors in the striatum. Furthermore, protein kinase C-dependent phosphorylation was shown to reduce the efficiency with which inhibitor-1 serves as a substrate for cAMP-dependent protein kinase in vitro and in vivo. 4) Finally, protein kinase C activation was shown to decrease the level of phosphorylation of cyclin-dependent kinase 5 substrates in the striatum, suggesting a possible role for protein kinase C in regulating cyclin-dependent kinase 5 activity.Item Identification and Characterization of a NEF-Associated Kinase(2004-05-04) Arora, Vivek Kumar; Bennett, MichaelThe nef gene encoded by primate lentiviruses is a major determinant of virulence in vivo. It is expressed early in the viral life cycle; its importance likely stems from its ability to prime the host environment for efficient viral replication. Reasonable models by which cellular phenotypes associated with Nef expression could enhance viral replication in vivo have been proposed. The molecular mechanisms by which Nef executes its functions, however, are poorly understood. The work presented here investigates the regulation of cellular proteins by Nef. I first identified a previously described Nef-associated kinase as the cellular kinase p21 activated kinase 2 (Pak2). This was done using proteolytic digestion of the Nef-associated kinase in multiple systems as well as by demonstrating the presence of active, ectopically expressed Pak2 associated with Nef in a cellular expression system. I further demonstrated that Nef induces Pak2 activation in vivo using multiple systems. First, Nef dependent activation of ectopically expressed tagged Pak2 was demonstrated in vivo. Second, an in gel renaturable kinase activity assay showed in cell extracts a Nef dependent kinase activity I subsequently demonstrated to be Pak2 by proteolytic digestion. Third, I showed that in vivo Nef expression induces the phosphorylation of Merlin at S518, a known and specific Pak2 substrate. The mechanism by which Nef leads to Pak2 activation was also addressed. Rho family GTPases are well-described endogenous activators of Pak2. Inhibition of Rho family GTPase activity in vivo also blocked Nef mediated activation of Pak2 as did mutation of the Rho GTPase binding site in Pak2. Thus, Nef induced Pak2 activation is dependent on endogenous Rho family GTPases. No Nef dependent effect on Rho GTPase activity levels, however, was detected. Instead, biochemical separation and cellular localization suggested that Nef mediates Pak2 activation by recruiting Pak2 to membranes where it encounters high concentrations of constituitively active Rho family GTPases. In summary, this work conclusively demonstrated that Pak2 associates with Nef and is activated by Nef via a endogenous Rho GTPase. Lastly, the potential roles of these molecular events in mediating Nef's pathogenic effects are discussed.Item Novel Activities of Kinase-Fold Enzymes from Legionella pneumophila(2020-08-01T05:00:00.000Z) Black, Miles; Cobb, Melanie H.; Tagliabracci, Vincent S.; Mendell, Joshua T.; Olson, Eric N.Protein kinases are fundamental mediators of cell signaling that transfer phosphate from ATP to their substrates. The protein kinase superfamily encompasses a vast and diverse trove of enzymes from all domains of life, including remote members that are barely recognizable by their primary amino acid sequence. SidJ (Substrate of Icm/Dot J) is a distant protein kinase homolog from the human pathogen Legionella pneumophila. Contamination of water supplies with Legionella bacteria is a frequent cause of deadly pneumonia outbreaks (Legionnaire's disease). SidJ is a secreted Legionella virulence factor required for bacterial intracellular replication, but it is unknown how SidJ contributes to pathogenesis of Legionnaire's disease, or if SidJ has maintained the kinase fold or catalytic activity. In this work, I determine that SidJ is a calmodulin-binding protein which adopts a protein kinase fold. However, instead of phosphorylation, it catalyzes protein polyglutamylation. SidJ utilizes ATP to form an isopeptide bond between the amino group of free glutamate and the 𝛾-carboxyl group of a glutamate of its substrate. During infection, SidJ polyglutamylates and inactivates a family of Legionella "all-in-one" ubiquitin ligases. Polyglutamylation is crucial step in the intracellular lifecycle of the bacterium and is required for full Legionella virulence in a eukaryotic host. SidJ reveals the unexpected catalytic versatility of the protein kinase fold, and highlights a unique strategy that pathogenic bacteria use to thrive within host cells. Interestingly, SidJ lacks key catalytic residues believed to be required for kinase activity. The discovery that SidJ is a polyglutamylating enzyme suggests that catalytically incompetent or 'pseudo' enzymes may lack activity only when assayed for the wrong reaction.Item The Role of Pyruvate Dehydrogenase in Cell Growth(2014-07-28) Rajagopalan, Kartik N.; Amatruda, James F.; DeBerardinis, Ralph J.; Brown, Michael S.; Lum, LawrenceOtto Warburg's observation that tumor cells have increased rates of glucose uptake and lactate secretion in comparison to normal cells spawned his notion that tumors have dysfunctional mitochondria. However, in addition to metabolizing glucose to lactate, tumors in vivo exhibit mitochondrial glucose oxidation, indicating activity of pyruvate dehydrognase (PDH), which gates entry of glucose derived carbon into the tricarboxylic acid (TCA) cycle. To test whether cells require glucose oxidation for proliferation, the work in this thesis establishes a model wherein PDH activity is suppressed using RNA interference. Small hairpin RNAs against the transcript encoding the PDHE1α protein were cloned into a retroviral vector which allowed doxycycline-inducible control of expression. Metabolism of cancer cells was studied in vitro using a combination of metabolomics and metabolic flux analysis. Growth in monolayer culture was performed in medium containing lipid-replete serum as well as serum in which lipids had been depleted. As expected, suppression of PDH activity reduced flow of carbon from glucose to the TCA cycle and to de novo fatty acid synthesis. Surprisingly, H460 lung cancer cells could tolerate a 60% reduction of PDH flux without any significant effect on proliferation rate, as long as lipids were present in the medium. Further examination of the effects of PDH silencing on the overall network of central carbon metabolism revealed enhanced channeling of carbon from glutamine to fatty acids and an increase in scavenging free fatty acids. Lipid depletion caused a reduction in growth rate of PDH deficient cells, and this defect was completely rescued by supplying free fatty acids to the medium. Together the data indicate that proliferating cells exhibit PDH activity that allows transfer of glucose carbon to citrate and the TCA cycle as well as ultimately into fatty acids. Importantly, suppression of PDH activity limits growth in conditions in which cancer cells do not have access to extracellular lipids. This work illustrates that compensatory pathways that sustain cell proliferation are activated during suppression of mitochondrial oxidation of glucose.Item Signals and Sensory Mechanisms that Impact Campylobacter jejuni-Host Interactions(2015-05-21) Luethy, Paul Michael; Sperandio, Vanessa; Hendrixson, David R.; Winter, Sebastian E.; Michael, AnthonyCampylobacter jejuni is a leading cause of bacterial diarrheal disease worldwide and a frequent commensal organism of the intestinal tract of poultry and other agriculturally-important animals. Upon infection of the avian host, C. jejuni likely responds to external stimuli present within the intestinal tract to establish commensalism. The sensing mechanisms and subsequent physiological responses by C. jejuni can be crucial for initial growth and colonization and long-term persistence within the infected host. However, how many of the signals and sensing mechanisms affecting C. jejuni biology are not fully understood. In this work, I explored signal transduction mechanisms and possible in vivo signals that may influence the colonization capacity of C. jejuni. One method C. jejuni employs to monitor environmental stimuli are two-component regulatory systems (TCSs). I analyzed the potential of C. jejuni Cjj81176_1484 (Cjj1484) and Cjj81176_1483 (Cjj1483) to encode a cognate TCS that influences expression of genes possibly important for C. jejuni growth and colonization. Through transcriptome analysis, I discovered that the regulons of the Cjj1484 histidine kinase and the Cjj1483 response regulator contain many common genes, which suggests these proteins likely form a cognate TCS. I found that this TCS generally functions to repress expression of specific proteins with roles in metabolism, iron/heme acquisition, and respiration. Furthermore, the TCS repressed expression of Cjj81176_0438 and Cjj81176_0439, which had previously been found to encode a gluconate dehydrogenase complex required for commensal colonization of the chick intestinal tract. However, the TCS and other specific genes whose expression is repressed by the TCS were not required for colonization of chicks. I observed that the Cjj1483 response regulator binds target promoters both in unphosphorylated and phosphorylated forms and influences expression of some specific genes independently of the Cjj1484 histidine kinase. I propose that this TCS may sense signals found in the host intestinal tract, wherein repression of genes may be relieved. In addition to characterizing the Cjj1484/Cjj1483 TCS, I explored the role of metabolites that are commonly found in the intestines -- organic acids and short chain fatty acids (SCFAs) -- in C. jejuni commensal colonization. C. jejuni has both acetate and lactate utilization pathways, as well as for acetate production. I observed that acetogenesis mutants incapable of producing acetate were deficient for colonization of the avian intestinal tract early during infection, but not at later points during infection. Furthermore, I found that an acetogenesis mutant was impaired during growth in a defined media containing solely amino acids and organic acids as carbon sources. Transcriptome analysis of the acetogenesis mutant identified the SCFA-induced regulon which contains metabolically important genes, many of which have been implicated in C. jejuni colonization and virulence. In addition, I found that peb1C, which was downregulated in the acetogenesis mutant, was important for colonization of the chick ceca. I further confirmed in vitro that physiological concentrations of the SCFAs acetate and butyrate activated expression of the SCFA-induced regulon whereas the organic acid lactate repressed these genes. I found that in vivo expression of the SCFA-induced regulon was highest in regions of the intestinal tract where SCFAs are present in the greatest concentration. Furthermore, butyrate counteracted the inhibitory effects of lactate when the two compounds were combined in culture in vitro. I propose that C. jejuni senses the concentration of SCFAs and organic acids to discriminate between different regions of the intestinal tract and to coordinate expression of colonization genes in the preferred niche for colonization. In effect, SCFA sensing and signaling allows C. jejuni to home to appropriate sites of the host for colonization and long-term persistence.Item Structural Insights into Sporulation in Bacillus sibtilis(2010-05-14) Lee, James; Gardner, Kevin H.PAS domains are modular domains that providing specific interaction surfaces for a diverse array of ligands, from small organic compounds to intra and intermolecular protein domains. As such, they are ubiquitous throughout signal transduction pathways in all three kingdoms of life: Bacteria, Archae, and Eucarya (Huang, Edery et al. 1993). The significant role played by PAS domains in Bacillus subtilis sporulation is underscored by the finding that the PAS-A domain is necessary for efficient phosphorylation in the sporulation kinase, KinA(Wang, Fabret et al. 2001). This activity is necessary for initiating a phosphorelay that results in the upregulation of genes required for sporulation. In KinA, dimerization is necessary for individual monomers of the histidine kinase domain to transphosphorylate partner subunits. The dimerization of KinA involves interactions in the PAS-A domain, but molecular details regarding PAS-A dimerization and its importance to KinA activity has not been previous characterized. To investigate these interactions within the context of the KinA homodimer, we expressed the N-terminal PAS-A domain and solved the X-ray crystal structure. Conformational variability was implicated through the observation of different orientations of the dimerization interface in two distinct structural models found in the asymmetric unit of the crystal lattice. These models were used to identify key interfacial residues and the roles of these were tested in a variety of ways by site-directed mutagenesis.Item Studies Toward the Syntheses of Antiarrythmic, Anti-Virulence, and Anticancer Small Molecules(2015-11-23) Adebesin, Adeniyi Michael; Kürti, László; Falck, John R.; Chen, Chuo; Corey, David R.This work is comprised of three projects: a) the development of antiarrythmic analogs of 17(R),18(S)-epoxyeicosatetraenoic acid for the treatment of atrial fibrillation, b) the development of potent inhibitors of QseC mediated virulence gene expression, and c) studies toward a biomimetic total synthesis of nigricanoside A. Antiarrhythmic analogs of 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EEQ): Arrhythmias such as atrial and ventricular fibrillation are a leading cause of death in the United States of America. However, the available drugs for the treatment of these deadly conditions can paradoxically induce proarrhythmic effects, amongst other side effects, and are individually insufficient for treatment. Through a neonatal rat cardiomyocyte assay, 17(R),18(S)-EEQ was found to possess negative chronotropic effects, a characteristic of antiarrhythmic activity. My work on this project led to the development of potent and metabolically robust analogs of 17(R),18(S)-EEQ, which are currently being developed by OMEICOS Therapeutics GmbH, an early stage drug development company, for the treatment of atrial fibrillation. Inhibitors of QseC mediated virulence gene expression: Quorum sensing E. coli regulator C (QseC), a membrane-bound histidine sensor kinase, mediates the expression of various virulence genes in Gram-negative bacteria such as Escherichia coli (EHEC), Salmonella typhimurium, and Francisella tularensis which are pathogenic to humans. Therefore, QseC is a potential target of anti-virulence antibacterial strategies. In collaboration with the Sperandio laboratory, my work on this project led to the development of novel inhibitors of QseC mediated virulence gene expression. Some of the analogs synthesized in this project are currently being investigated by GlaxoSmithKline as anti-virulence agents. Studies toward a biomimetic total synthesis of nigricanoside A: Nigricanoside A, a novel ether-linked glycoglycerolipid with 7 unassigned stereocenters, was reported to possess potent (IC50 ≈ 3 nM) antimitotic activity against MCF-7 and HCT-116 cancer cell lines. However, the rarity of the natural product precluded further structural and biological studies. With the aid of biomimetic hypotheses and literature precedents, the Falck laboratory reduced the stereochemical uncertainty associated with the structure elucidation of the nigricanosides. Furthermore, one of the biomimetic hypotheses inspired the development of a novel stereocontrolled distal epoxidation of conjugated dienols. Armed with this methodology, my work on this project led to the synthesis of the three major fragments of a nigricanoside.Item Thioredoxin-1 Inhibition Promotes MLKL Activation: Biochemical Insights Into a Suppressor of the Necroptotic Pathway(2017-07-13) Reynoso, Eduardo; Amatruda, James F.; Wang, Zhigao; Abrams, John M.; Chen, Zhijian J.Necroptosis is an immunogenic caspase-independent cell death program. While mainly serving to defend against viral infection, dysregulated necroptotic signaling contributes to the pathology of a growing number of human diseases associated with inflammation, neurodegeneration, and ischemic injuries. Since its emergence, many key components of the mammalian necroptotic pathway have been discovered including RIP1/3 kinases and MLKL, which together, form the core of the necrosome complex. RIP3-dependent phosphorylation of MLKL promotes the formation of high molecular weight MLKL polymers that disrupt the integrity of the plasma membrane leading to cell death. However, the mechanistic details of MLKL polymerization and its precise function in executing necroptosis remain poorly understood. To gain insight into this process, the crosslinking property of the specific MLKL inhibitor Necrosulfonamide (NSA) was exploited as a strategy for identifying novel MLKL interacting proteins. Immunoprecipitation of NSA crosslinked MLKL complexes followed by mass spectrometric analysis revealed Thioredoxin-1, an essential cytosolic thiol oxidoreductase, to be a potential MLKL regulator. Based on its ability to reduce disulfide bonds on specific protein targets, it was hypothesized that Thioredoxin-1 actively maintains MLKL in a reduced monomeric state to suppress the occurrence of spontaneous necroptosis. Therefore, genetic and pharmacological approaches were taken to perturb Thioredoxin-1 activity as a means for testing whether it would induce necroptosis in human cells. The data presented here demonstrates that inhibiting Thioreoxin-1 activity using the compound PX-12 promoted RIP3-dependent MLKL S358 phosphorylation, polymer formation, and caspase-independent necrotic cell death. Additionally, PX12-induced cell death was rescued by co-treating cells with NSA highlighting the involvement of MLKL in this cell death pathway. These results were corroborated by shRNA-mediated knockdown of Thioreoxin-1 mRNA. Altogether, these findings point to Thioredoxin-1 as a critical regulator of MLKL activity and necroptosis in human cells.Item With No Gly-Loop Family of Kinases(2019-04-11) Beraki, Tsebaot Ghebretinsae; Tagliabracci, Vincent S.; Whitehurst, Angelique Wright; Reese, Michael L.; Phillips, Margaret A.; Cobb, Melanie H.Toxoplasma gondii replicates within a protective organelle called the parasitophorous vacuole (PV). The PV is filled with a network of tubulated membranes, which are thought to facilitate trafficking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network's functions. Phosphoproteomics data indicated that many PV resident proteins are phosphorylated, including ones that have roles is the biogenesis and maintenance of these tubular networks. Since phosphorylation is a common method to regulate protein functions we hypothesized that PV resident kinases must regulate PV/IVN functions. Protein kinases are enzymes that modify proteins with phosphate molecules. Using bioinformatics, we identified an unusual family of kinases (WNG kinases) that lack a structural motif, called the Gly-loop, that is absolutely required for the activity of all previously described kinases. In this work, I show that the most conserved WNG kinase, WNG1 as well as WNG2 are catalytically active. The WNG kinases are only found in certain intracellular parasites, such as the human pathogen Toxoplasma gondii. I identify the parasite secreted kinase WNG1 as a critical regulator of tubular membrane biogenesis. By solving the crystal structure of a pseudokinase in the WNG1 family, I show members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, I find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, I show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, my results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.