Phg2 is a large (1387 aa) TKL-family serine/threonine protein kinase of Dictyostelium discoideum. An N-terminal domain binds PI(4,5)P2 and PI(4)P and recruits the protein to the plasma membrane, a central Ras-association (RA) domain binds active Rap1-GTP, an adjacent region binds the Adrm1 homolog, and a C-terminal kinase domain lies at residues 807-1072. Phg2 acts as a Rap1 effector at the cell cortex and leading edge, where it is needed for cell-substrate adhesion, particle binding during phagocytosis, actin organization at the substrate interface, cell motility and chemotaxis; it is required for chemoattractant-induced phosphorylation and disassembly of cortical myosin II at the leading edge, apparently acting upstream of myosin heavy chain kinase. Phg2 also acts cell-autonomously as a negative regulator of nutrient-controlled initiation of multicellular development, and promotes STATc activation during hyperosmotic stress, apparently by phosphorylating and inhibiting the tyrosine phosphatase PTP3 on Ser747.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000281 mitotic cytokinesis | IMP PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | UNDECIDED | Summary: IMP annotation to mitotic cytokinesis from the original phg2 mutant characterization. Reason: The results section of PMID:15194808 is not in the cached text, so the cytokinesis assay cannot be checked. An independent phg2-null strain was explicitly reported as not multinucleated, which argues against a strong cytokinesis defect, but strain backgrounds differ markedly for this gene. Cannot confirm or refute without the full text. Supporting Evidence: PMID:17371831 phg2-null cells in either HL5 axenic growth medium or phosphate buffer are very flat and spread compared with wild-type cells (and are not multinucleated) |
| GO:0000902 cell morphogenesis | IMP PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | KEEP AS NON CORE | Summary: phg2 null cells have altered cell shape (flat, spread, with extra lamellipodia/filopodia). Reason: Cell shape change is a downstream consequence of disturbed adhesion and actin/myosin regulation; reasonable phenotype-level annotation but not a core function. Supporting Evidence: PMID:17371831 phg2-null cells in either HL5 axenic growth medium or phosphate buffer are very flat and spread compared with wild-type cells (and are not multinucleated) |
| GO:0004672 protein kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of protein kinase activity for a TKL-family kinase with a canonical kinase domain. Reason: Consistent with the kinase domain and with experimental evidence of Ser/Thr kinase activity; the more specific term GO:0004674 is also present. Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain PMID:17371831 Gebbie et al. (2004) demonstrated that the expression of Phg2 in Escherichia coli led to the in vivo phosphorylation of bacterial proteins on Ser/Thr residues, indicating that Phg2 has Ser/Thr kinase activity |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro-based kinase activity; Phg2 has a complete protein kinase domain (807-1072). Reason: Correct, if general; GO:0004674 captures the specific activity. Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: Ser/Thr kinase activity shown by phosphorylation of bacterial proteins on Ser/Thr when Phg2 was expressed in E. coli. Reason: Experimental annotation by curators; supported by domain architecture. Purified recombinant Phg2 was inactive on exogenous substrates under tested conditions, so direct substrates remain undefined, but the activity assignment is sound. Supporting Evidence: PMID:17371831 Gebbie et al. (2004) demonstrated that the expression of Phg2 in Escherichia coli led to the in vivo phosphorylation of bacterial proteins on Ser/Thr residues, indicating that Phg2 has Ser/Thr kinase activity PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain |
| GO:0004674 protein serine/threonine kinase activity | IDA PMID:24587195 Identification of the protein kinases Pyk3 and Phg2 as regul... | ACCEPT | Summary: Phg2 is required for PTP3 Ser747 phosphorylation and binds PTP3 directly, consistent with Phg2 phosphorylating PTP3. Reason: Evidence is genetic plus direct binding rather than an in vitro kinase assay, but it is consistent with the established Ser/Thr kinase activity; defer to curator. Supporting Evidence: PMID:24587195 In contrast, Myc-PTP3 S747 phosphorylation was nearly completely abolished in the phg2− strain PMID:24587195 The pull-down results demonstrate a direct interaction between Myc-PTP3 purified from phg2− cells and recombinant GST-Phg2 |
| GO:0004674 protein serine/threonine kinase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated Ser/Thr kinase activity assignment (EC 2.7.11.1). Reason: Consistent with experimental data. Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain |
| GO:0005515 protein binding | IPI PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | MODIFY | Summary: IPI with RasG (P15064), a small GTPase, via the Ras-binding domain. Reason: protein binding is uninformative; the interaction is with a Ras-family small GTPase through the RA/RBD domain, captured by small GTPase binding. Note later work found the RA domain preferentially binds Rap1-GTP over Ras-GTP. Proposed replacements: small GTPase binding Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain PMID:17371831 We confirmed that the Phg2 RA domain preferentially binds Rap1-GTP over Ras-GTP |
| GO:0005515 protein binding | IPI PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | MODIFY | Summary: IPI with RasS (P32254), a small GTPase, via the Ras-binding domain. Reason: protein binding is uninformative; small GTPase binding describes the RBD-mediated interaction. Rap1 appears to be the preferred partner in vivo. Proposed replacements: small GTPase binding Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain PMID:17371831 We confirmed that the Phg2 RA domain preferentially binds Rap1-GTP over Ras-GTP |
| GO:0005515 protein binding | IPI PMID:16987957 A Phg2-Adrm1 pathway participates in the nutrient-controlled... | REMOVE | Summary: Phg2 core region (residues 668-755) binds Adrm1 (yeast two-hybrid and GST pull-down). Reason: Bare protein binding carries no functional information and no more informative MF term fits (Adrm1 is a proteasomal ubiquitin receptor homolog with no defined biochemical role in this pathway). Removal does not dispute the interaction, which is well supported by yeast two-hybrid and GST pull-down and is functionally linked to development initiation. Supporting Evidence: PMID:16987957 the Phg2 core region interacted with the Dictyostelium adhesion-regulating molecule (Adrm1) protein |
| GO:0005515 protein binding | IPI PMID:24587195 Identification of the protein kinases Pyk3 and Phg2 as regul... | MODIFY | Summary: Direct binding of Phg2 to the tyrosine phosphatase PTP3 in reciprocal pull-downs. Reason: PTP3 is a phosphatase that Phg2 appears to inhibit by phosphorylation; phosphatase binding is more informative than protein binding. Proposed replacements: phosphatase binding Supporting Evidence: PMID:24587195 The pull-down results demonstrate a direct interaction between Myc-PTP3 purified from phg2− cells and recombinant GST-Phg2 PMID:24587195 We present evidence that both TKL proteins modulate STATc-signalling and that Phg2 but not Pyk3 acts as a PTP3 inhibitor. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: ATP binding inferred from the protein kinase ATP-binding site. Reason: Required for kinase activity; correct. Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain |
| GO:0005546 phosphatidylinositol-4,5-bisphosphate binding | IDA PMID:16325504 A novel phosphatidylinositol 4,5-bisphosphate-binding domain... | ACCEPT | Summary: N-terminal domain (81-193) binds PI(4,5)P2 and PI(4)P and targets Phg2 to the plasma membrane. Reason: Direct lipid-binding evidence; membrane recruitment via this domain is essential for function in phagocytosis. Supporting Evidence: PMID:16325504 we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate PMID:16325504 Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Phylogenetically inferred cytoplasmic activity. Reason: Phg2 has a cytosolic pool in unstimulated cells but its functional site is the plasma membrane/cell cortex; cytoplasm is correct but not the informative location. Supporting Evidence: PMID:17371831 Upon chemoattractant stimulation, GFP-Phg2 rapidly and transiently translocates to the cell cortex |
| GO:0005886 plasma membrane | IDA PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: Phg2 localizes to the plasma membrane. Reason: Supported by membrane targeting through the PI(4,5)P2-binding domain and cortical localization of GFP-Phg2. Supporting Evidence: PMID:16325504 Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process PMID:17371831 Upon chemoattractant stimulation, GFP-Phg2 rapidly and transiently translocates to the cell cortex |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular location mapping to plasma membrane. Reason: Consistent with experimental data. Supporting Evidence: PMID:16325504 Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process |
| GO:0005938 cell cortex | IDA PMID:17371831 Rap1 controls cell adhesion and cell motility through the re... | ACCEPT | Summary: GFP-Phg2 translocates to the cell cortex upon chemoattractant stimulation, colocalizing with Rap1-GTP. Reason: Direct imaging evidence. Supporting Evidence: PMID:17371831 Upon chemoattractant stimulation, GFP-Phg2 rapidly and transiently translocates to the cell cortex |
| GO:0006909 phagocytosis | IMP PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: phg2 mutants were isolated in a screen for phagocytosis defects and are defective in particle binding. Reason: Phagocytosis defect confirmed independently and depends on membrane targeting of Phg2; adhesion-step role in phagocytosis is a core function. Supporting Evidence: PMID:15194808 Whereas these proteins share a common role in cellular adhesion and phagocytosis, Phg2 plays a unique role in the modeling of actin-rich focal sites and in cell motility PMID:16325504 Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process |
| GO:0006935 chemotaxis | IMP PMID:17371831 Rap1 controls cell adhesion and cell motility through the re... | ACCEPT | Summary: phg2-null cells chemotax poorly, with broad leading edges, lateral pseudopodia and reduced chemoattractant-induced F-actin polymerization. Reason: Complemented phenotype; Phg2 acts downstream of Rap1 at the leading edge to regulate myosin II disassembly during chemotaxis. Supporting Evidence: PMID:17371831 have a broader leading edge than wild-type cells, and produce numerous dominant lateral pseudopodia PMID:17371831 Phg2 localizes to the leading edge of polarized chemotaxing cells |
| GO:0007015 actin filament organization | IMP PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: Phg2 is required for normal actin organization, particularly at the cell-substrate interface. Reason: Directly stated phenotype in primary characterization. Supporting Evidence: PMID:15194808 Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton PMID:15194808 phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility |
| GO:0007165 signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of signal transduction. Reason: Phg2 is a Rap1 effector kinase relaying Rap1-GTP signals to the cytoskeleton; general but correct. Supporting Evidence: PMID:16769729 Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain |
| GO:0009653 anatomical structure morphogenesis | IEA GO_REF:0000117 | REMOVE | Summary: ARBA machine-learning annotation to anatomical structure morphogenesis. Reason: Phg2 has no demonstrated role in morphogenesis of a multicellular structure; once development starts, phg2 mutants develop with wild-type morphology. Cell-level shape effects are already covered by cell morphogenesis. Supporting Evidence: PMID:16987957 development of starved phg2 mutant cells was morphologically identical to that of wild-type cells |
| GO:0009967 positive regulation of signal transduction | IEA GO_REF:0000117 | KEEP AS NON CORE | Summary: ARBA annotation to positive regulation of signal transduction. Reason: Loosely consistent with Phg2 promoting STATc signaling by inhibiting PTP3, but general; non-core. Supporting Evidence: PMID:24587195 We present evidence that both TKL proteins modulate STATc-signalling and that Phg2 but not Pyk3 acts as a PTP3 inhibitor. |
| GO:0010628 positive regulation of gene expression | IMP PMID:24587195 Identification of the protein kinases Pyk3 and Phg2 as regul... | KEEP AS NON CORE | Summary: Phg2 is required for induction of STATc-dependent genes in response to hyperosmotic stress. Reason: Indirect effect via STATc signaling; valid but non-core. Supporting Evidence: PMID:24587195 We present evidence that both TKL proteins modulate STATc-signalling and that Phg2 but not Pyk3 acts as a PTP3 inhibitor. |
| GO:0019899 enzyme binding | IEA GO_REF:0000117 | MARK AS OVER ANNOTATED | Summary: ARBA annotation to enzyme binding. Reason: Uninformative; specific binding partners are better captured by small GTPase binding (Rap1) and phosphatase binding (PTP3). Supporting Evidence: PMID:16769729 Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain |
| GO:0030666 endocytic vesicle membrane | IDA PMID:16325504 A novel phosphatidylinositol 4,5-bisphosphate-binding domain... | KEEP AS NON CORE | Summary: The PI(4,5)P2-binding N-terminal domain of Phg2 (GFP-Nt-Phg2) labels macropinosome/phagosome membranes until closure. Reason: Abstract-only access; evidence relates to the isolated lipid-binding domain used as a PI(4,5)P2 probe, and it leaves the vesicle at closure. Defer to curator but treat as non-core. Supporting Evidence: PMID:16325504 GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure |
| GO:0031156 regulation of sorocarp development | IMP PMID:16987957 A Phg2-Adrm1 pathway participates in the nutrient-controlled... | ACCEPT | Summary: phg2 mutants initiate development in the presence of nutrients; Phg2 is a cell-autonomous negative regulator of development initiation. Reason: Well supported by full-text data including complementation requiring the kinase domain. Supporting Evidence: PMID:16987957 Phg2 must act as a negative regulator of development initiation |
| GO:0031252 cell leading edge | IDA PMID:17371831 Rap1 controls cell adhesion and cell motility through the re... | ACCEPT | Summary: GFP-Phg2 localizes to the leading edge of chemotaxing cells, as does Rap1-GTP. Reason: Direct imaging evidence. Supporting Evidence: PMID:17371831 Phg2 localizes to the leading edge of polarized chemotaxing cells |
| GO:0031267 small GTPase binding | IPI PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: Phg2 binds Rap1 (P18613) via its Ras-association domain. Reason: Independently confirmed; RA domain preferentially binds Rap1-GTP and is required for Phg2 function. Supporting Evidence: PMID:16769729 Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain PMID:17371831 We confirmed that the Phg2 RA domain preferentially binds Rap1-GTP over Ras-GTP |
| GO:0031589 cell-substrate adhesion | IMP PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: phg2 mutant was isolated as adhesion-defective. Reason: Core phenotype; independently confirmed though direction differs between strain backgrounds. Supporting Evidence: PMID:15194808 Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion |
| GO:0031589 cell-substrate adhesion | IMP PMID:17371831 Rap1 controls cell adhesion and cell motility through the re... | ACCEPT | Summary: Independent phg2-null strain (KAx-3) shows strongly altered substratum attachment, complemented by tagged Phg2. Reason: Confirms Phg2 is required for normal regulation of cell-substrate adhesion downstream of Rap1. Supporting Evidence: PMID:17371831 Our phg2-null strain exhibited a very strong increase in cell attachment compared with our parental wild-type cells |
| GO:0031670 cellular response to nutrient | IMP PMID:16987957 A Phg2-Adrm1 pathway participates in the nutrient-controlled... | ACCEPT | Summary: phg2 mutant cells fail to respond normally to nutrients when deciding to initiate development. Reason: Direct conclusion of the paper; not secondary to adhesion defect. Supporting Evidence: PMID:16987957 resulted rather from a defect in the cellular response to nutrients |
| GO:0048870 cell motility | IMP PMID:15194808 Phg2, a kinase involved in adhesion and focal site modeling ... | ACCEPT | Summary: phg2 mutant cells are defective in cell motility. Reason: A phenotype specific to phg2 among the adhesion mutants compared. Supporting Evidence: PMID:15194808 phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility |
| GO:0051489 regulation of filopodium assembly | IMP PMID:17371831 Rap1 controls cell adhesion and cell motility through the re... | KEEP AS NON CORE | Summary: phg2-null cells have numerous and more prominent filopodia-like structures. Reason: Morphological phenotype downstream of cytoskeletal misregulation; non-core. Supporting Evidence: PMID:17371831 The cells often have numerous and more prominent filipodia-like structures compared with wild-type cells |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-based protein serine kinase activity. Reason: Consistent with Ser/Thr kinase activity (EC 2.7.11.1). Supporting Evidence: PMID:15194808 PHG2 encodes a novel serine/threonine kinase with a ras-binding domain |
| GO:1904894 positive regulation of receptor signaling pathway via STAT | IMP PMID:24587195 Identification of the protein kinases Pyk3 and Phg2 as regul... | KEEP AS NON CORE | Summary: Phg2 promotes STATc phosphorylation/activation in response to hyperosmotic stress, likely by inhibiting PTP3. Reason: Experimentally supported but a secondary signalling role relative to adhesion/motility. Supporting Evidence: PMID:24587195 We present evidence that both TKL proteins modulate STATc-signalling and that Phg2 but not Pyk3 acts as a PTP3 inhibitor. PMID:24587195 In contrast, Myc-PTP3 S747 phosphorylation was nearly completely abolished in the phg2− strain |
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Download this section (compressed HTML)Q: Does Phg2 directly phosphorylate PTP3 Ser747 and/or myosin II heavy chain (or MHCK-A), or does it act through an intermediate kinase? Recombinant Phg2 has so far been inactive on exogenous substrates.
Q: Why do phg2-null strains in DH1 and KAx-3 backgrounds show opposite adhesion phenotypes (reduced vs increased attachment)?
Q: Would GO:0110014 (negative regulation of aggregation involved in sorocarp development) or a term for negative regulation of initiation of development better capture the phg2 developmental phenotype than regulation of sorocarp development?
Q: Does Phg2 genuinely bind RasG and RasS in vivo, given that its RA domain preferentially binds Rap1-GTP?
Experiment: In vitro kinase assays with Phg2 purified from Dictyostelium (with and without co-purified Rap1-GTP) on recombinant PTP3 and myosin II tail fragments, plus a kinase-dead (catalytic Lys/Asp mutant) Phg2 complementation test of adhesion, chemotaxis and development phenotypes.
Experiment: Phosphoproteomic comparison of wild-type and phg2-null cells after cAMP stimulation and hyperosmotic stress to identify direct Phg2 substrates.
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