Bradyzoite Pseudokinase 1 Is Crucial for Efficient Oral Infectivity of the Toxoplasma gondii Tissue Cyst

Buchholz, Kerry R.; Bowyer, Paul W.; Boothroyd, John C.

doi:10.1128/ec.00343-12

Cited by 48 publications

(42 citation statements)

References 42 publications

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“…We also examined the proteins Ror1 (receptor tyrosine kinase-like orphan receptor 1), BubR1 [also known as BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B)] and RYK (receptor-like tyrosine kinase), which despite containing the key residues of the VAIK, HRD and DFG motifs did not exhibit catalytic activity in earlier studies [33–36]. In addition to the 23 human and two mouse pseudokinases chosen for examination, we also analysed the bacterial pseudokinase MviN [28], two plant pseudokinases, CRN (CORYNE) [37] and TARK1 (tomato atypical receptor-like kinase 1) [38], and three Toxoplasma gondii pseudokinases, ROP5B I [29], ROP2 [39] and BPK1 [40]. …”

Section: Resultsmentioning

confidence: 99%

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Murphy

Zhang

Young

et al. 2013

Biochemical Journal

254

319

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Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

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Section: Resultsmentioning

confidence: 99%

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Murphy

Zhang

Young

et al. 2013

Biochemical Journal

254

319

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“…By profiling FACS-enriched differentiated populations, we captured transcriptional differences between tachyzoites and bradyzoites with greater sensitivity and dynamic range than achieved by previous datasets (Behnke et al, 2008;Buchholz et al, 2013;Lescault et al, 2010;Naguleswaran et al, 2010;Pittman et al, 2014;Radke et al, 2005;Ramakrishnan et al, 2019). These changes likely reflect a combination of factors including altered replication, nutrient availability, and general stress responses, in addition to the bradyzoite differentiation program.…”

Section: Discussionmentioning

confidence: 99%

Identification of a master regulator of differentiation inToxoplasma

Waldman

Schwarz

Wadsworth

et al. 2019

Preprint

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Toxoplasma gondii chronically infects a quarter of the world's population, and its recrudescence can cause life-threatening disease in immunocompromised individuals and recurrent ocular lesions in the immunocompetent. Chronic stages are established by differentiation of rapidly replicating tachyzoites into slow-growing bradyzoites, which form intracellular cysts resistant to immune clearance and existing therapies. Despite its central role in infection, the molecular basis of chronic differentiation is not understood. Through Cas9-mediated genetic screening and single-cell transcriptional profiling, we identify and characterize a putative transcription factor (BFD1) as necessary and sufficient for differentiation. Translation of BFD1 appears to be stress regulated, and its constitutive expression elicits differentiation in the absence of stress. As a Myb-like factor, BFD1 provides a counterpoint to the ApiAP2 factors which dominate our current view of parasite gene regulation. Overall, BFD1 provides a genetic switch to study and control Toxoplasma differentiation, and will inform prevention and treatment of chronic infection.

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“…We were surprised to find that a small family of parasite kinases appear to completely lack the glycine-rich, or P-loop, that is found in all canonical kinases and is required for binding the ATP in the active site (28,29) (Supplemental Fig S1a,b). These kinases include three proteins annotated as ROPKs (ROP33, ROP34, and ROP35), and a pseudokinase, BPK1, that has previously been identified as PV resident and a component of the bradyzoite cyst wall (30). Phylogenetic analysis 3 gave clear support for these proteins forming a clade that is distinct from canonical protein kinases (Figure 1), including the parasite ROPKs.…”

Section: Identification Of a Divergent Family Of Coccidian Secreted Kmentioning

confidence: 92%

“…As noted above, BPK1 has previously been identified as a PV-resident pseudokinase (30). We thus sought to assess the localization of other WNG kinases, and concentrated on the most divergent members of the family in Toxoplasma: ROP34 and ROP35 ( Figure 1).…”

Section: Wng Kinases Are Secreted Into the Parasitophorous Vacuolementioning

confidence: 99%

A divergent kinase lacking the glycine-rich loop regulates membrane ultrastructure of theToxoplasmaparasitophorous vacuole

Beraki¹,

Hu²,

Broncel³

et al. 2018

Preprint

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Apicomplexan parasites replicate within a protective organelle called the parasitophorous vacuole (PV). The Toxoplasma gondii 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. Here, we identify the parasite secreted kinase WNG1 as a critical regulator of tubular membrane biogenesis. WNG1 family members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, we 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, we 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, our results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV formation during parasite infection.

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Bradyzoite Pseudokinase 1 Is Crucial for Efficient Oral Infectivity of the Toxoplasma gondii Tissue Cyst

Cited by 48 publications

References 42 publications

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Identification of a master regulator of differentiation inToxoplasma

A divergent kinase lacking the glycine-rich loop regulates membrane ultrastructure of theToxoplasmaparasitophorous vacuole

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