ppkA (PA0074) encodes PpkA, a Hanks-type serine/threonine protein kinase with a type II inner-membrane topology. It has an N-terminal cytoplasmic kinase domain, a single transmembrane segment, and a periplasmic region containing a von Willebrand factor A domain. PpkA is encoded in the Hcp secretion island I (HSI-I) and post-translationally activates the H1 type VI secretion system (H1-T6SS). On a signal that is still unknown, its periplasmic domains dimerise and it autophosphorylates on Thr158 and Thr161 of the activation loop; it then phosphorylates the FHA-domain scaffold protein Fha1 on Thr362. Phosphorylated Fha1 recruits the Fha1-ClpV1 complex to the T6SS apparatus and triggers Hcp1 secretion. The PP2C phosphatase PppA (PA0075) reverses the phosphorylation, and the periplasmic protein TagR acts upstream to promote PpkA activity. PpkA also has a structural role in T6SS assembly. It is induced during infection and is required for full virulence in neutropenic mice.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000166 nucleotide binding | IEA GO_REF:0000104 | TOO GENERAL | Summary: Nucleotide binding from the kinase-domain keyword mapping. Reason: Entailed by the ATP binding row kept in this review. |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: Protein kinase activity from the InterPro kinase-domain mapping. Reason: Entailed by the accepted protein serine/threonine kinase activity (GO:0004674 is_a GO:0004672). |
| GO:0004674 protein serine/threonine kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Ser/Thr kinase IBA from PTN001981567, a large Hanks-kinase node with 17 seeds (mostly M. tuberculosis Pkn kinases, B. subtilis PrkC and several human kinases). Reason: PpkA has an intact Hanks kinase domain (active site D129) and phosphorylates threonines on itself and on Fha1. The node is seeded mainly by experimentally characterised bacterial Ser/Thr kinases, so the transfer is sound. PpkA appearing among the sources reflects its own (PseudoCAP) annotation. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN001981567 · PTN001981567 SUPPORTS TRANSFER Hanks Ser/Thr kinase node; 16 other seeds (M. tuberculosis PknA-L paralogs, B. subtilis PrkC O34507, human NEK6/NEK7/PBK/MAP3K20 and others) carry the activity, independently of PpkA. UniProtKB:Q9I758 · ppkA (self; PseudoCAP IDA PMID:9852028) SOURCE WEAK OR INFERRED The cited abstract reports an in vivo catalytic-site mutant phenotype, not a kinase assay (full text not read). The term itself is independently established. Supporting Evidence: PMID:19400797 Our prior work has shown that PpkA-catalyzed phosphorylation of Fha1 at T362 is essential for Hcp1 secretion. This reaction is efficiently catalyzed in vitro by the kinase domain of PpkA (Mougous et al., 2007). |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:9852028 A novel serine/threonine protein kinase homologue of Pseudom... | ACCEPT | Summary: PseudoCAP IDA, one of 17 seeds of the PTN001981567 IBD. The cited abstract shows only that overexpressed PpkA is toxic and that a catalytic-site substitution abolishes toxicity. Reason: The term is correct. PpkA autophosphorylates on Thr158/Thr161 and phosphorylates Fha1 Thr362, a reaction its kinase domain catalyses in vitro (PMID:19400797, citing Mougous et al. 2007). The cited paper is only abstract-available. Its abstract infers kinase function from an in vivo catalytic-site mutant ("suggesting that the kinase domain of PpkA is functional"), which reads as genetic rather than direct-assay evidence. The evidence code is questioned in a SUSPECTED defect; the annotation is kept. Supporting Evidence: PMID:9852028 However, a single amino acid change at the catalytic site of the kinase domain eliminated the toxic effect of PpkA on bacterial cells, suggesting that the kinase domain of PpkA is functional within bacterial cells. PMID:19400797 Our prior work has shown that PpkA-catalyzed phosphorylation of Fha1 at T362 is essential for Hcp1 secretion. This reaction is efficiently catalyzed in vitro by the kinase domain of PpkA (Mougous et al., 2007). |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Ser/Thr kinase from EC 2.7.11.1 / ARBA mapping; consistent. Reason: Same claim as the IBA and IDA rows; supported experimentally. Supporting Evidence: PMID:19400797 Our prior work has shown that PpkA-catalyzed phosphorylation of Fha1 at T362 is essential for Hcp1 secretion. This reaction is efficiently catalyzed in vitro by the kinase domain of PpkA (Mougous et al., 2007). |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: ATP binding of the Hanks kinase domain. Reason: True of an active protein kinase (Pkinase PF00069 with intact catalytic residues), but a substrate-binding term subsumed by the kinase activity. Supporting Evidence: file:PSEAE/ppkA/ppkA-uniprot.txt Pfam; PF00069; Pkinase; 1. |
| GO:0016301 kinase activity | IEA GO_REF:0000104 | TOO GENERAL | Summary: Generic kinase activity. Reason: Entailed by the accepted protein serine/threonine kinase activity. |
| GO:0016740 transferase activity | IEA GO_REF:0000104 | TOO GENERAL | Summary: Generic transferase activity. Reason: Entailed by the accepted protein serine/threonine kinase activity. |
| GO:0050714 positive regulation of protein secretion | IDA PMID:19400797 TagR promotes PpkA-catalysed type VI secretion activation in... | ACCEPT | Summary: PpkA activates H1-T6SS secretion of Hcp1. It autophosphorylates and phosphorylates Fha1, which triggers the system. Reason: Full text read. PpkA is a direct post-translational activator of the secretion apparatus (kinase on Fha1), with dimerisation-induced activation shown. GO has no "positive regulation of protein secretion by the type VI secretion system" term, so this is the most precise available term. PpkA's additional structural role in the apparatus is noted in the description. Supporting Evidence: PMID:19400797 Previously, we showed that Pseudomonas aeruginosa T6S is posttranslationally activated upon phosphorylation of Fha1, an FHA domain protein, by PpkA, a membrane-spanning threonine kinase. PMID:19400797 We found that only the cytoplasmic domains of PpkA are essential for Fha1 phosphorylation and Hcp1 secretion. |
| GO:0005886 plasma membrane | IC PMID:19400797 TagR promotes PpkA-catalysed type VI secretion activation in... | NEW | Summary: Inner (plasma) membrane location. PpkA is a type II membrane-spanning kinase with a cytoplasmic kinase domain and periplasmic domains whose dimerisation activates it. Reason: Two independent lines agree: a predicted single TM segment (residues 355-375), and functional data showing a cytoplasmic kinase domain with extracellular-side domains that sense the activating cue. In a Gram-negative bacterium this places PpkA in the inner membrane. Supporting Evidence: PMID:19400797 These proteins include PpkA, a type II membrane-spanning Hanks-type threonine kinase, PppA, a PP2C family protein phosphatase, and Fha1, a protein containing a Forkhead Associated (FHA) domain file:PSEAE/ppkA/ppkA-uniprot.txt TRANSMEM 355..375 |
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Experiment: Identify the activating signal by screening for conditions that raise PpkA Thr158/Thr161 phosphorylation and Fha1 Thr362 phosphorylation in vivo, and test direct TagR-PpkA binding with purified periplasmic domains.
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