Pyk3 (DPYK3, PkyA) is a large (1338 aa) tyrosine kinase-like (TKL) group protein kinase of Dictyostelium discoideum with a JAK-like tandem architecture: a long, partly disordered N-terminal region, a central pseudokinase domain (KII) and a C-terminal catalytic TKL domain (KI). Although Dictyostelium lacks orthodox tyrosine kinases and JAKs, the Pyk3 KI domain autophosphorylates on tyrosine and directly phosphorylates the transcription factor STATc on its activating residue Tyr922; the pseudokinase domain acts, like the JAK JH2 domain, as a negative regulator of the kinase domain. Autophosphorylated tyrosine(s) on Pyk3 provide a docking site for the STATc SH2 domain. Pyk3 activity is increased by hyperosmotic stress and cGMP, and Pyk3 acts redundantly with the related TKL kinase Pyk2 to activate STATc in the hyperosmotic stress response, promoting STATc nuclear accumulation and induction of STATc target genes. Pyk3 is cytosolic in resting cells and relocates to the actin-rich cell cortex under stress. Loss of Pyk3 alters STATc phosphorylation dynamics and prestalk/prespore patterning during multicellular development.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000117 | MODIFY | Summary: Correct but uninformative ARBA annotation; Pyk3 is an experimentally demonstrated protein tyrosine kinase. Reason: Catalytic activity is far too general; the specific activity (protein tyrosine kinase activity) is experimentally established. Proposed replacements: protein tyrosine kinase activity Supporting Evidence: PMID:25143406 In the construct GST-Pyk3-KI, where KI was expressed in isolation from KII, the TKL directed tyrosine phosphorylation of His-STATc on Y922. |
| GO:0004672 protein kinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of protein kinase activity for the TKL family node; consistent with the experimental data. Pyk3 itself (DDB_G0283699) and its paralog Pyk2 (DDB_G0285321) are among the experimental sources, which is expected. Reason: Pyk3 has a catalytically competent C-terminal TKL domain whose activity depends on the conserved ATP-site lysine K1084; the general kinase term is correct. Supporting Evidence: PMID:25143406 Tyrosine phosphorylation of Pyk3 was ablated when K1084, in the predicted site of ATP binding |
| GO:0004672 protein kinase activity | IDA PMID:18657170 Dictyostelium kinase DPYK3 negatively regulates STATc signal... | ACCEPT | Summary: Experimental annotation from Lee et al. 2008 (abstract-only in cache; the kinase assay is presumably in the full text). Protein kinase activity of Pyk3 is well established independently. Reason: The function is clearly correct for Pyk3 (autophosphorylation and STATc phosphorylation by recombinant Pyk3 in PMID:25143406 and PMID:8898113); defer to the curator. The more specific GO:0004713 is also annotated. Supporting Evidence: PMID:18657170 DPYK3, a member of the Dictyostelium TKL (tyrosine kinase like) kinase family, was ablated by homologous recombination. PMID:25143406 Tyrosine phosphorylation of Pyk3 was ablated when K1084, in the predicted site of ATP binding |
| GO:0004672 protein kinase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO inference from the Ser-Thr/Tyr kinase catalytic domain and tyrosine kinase active-site signatures; correct. Reason: Consistent with experimental evidence for Pyk3 kinase activity. Supporting Evidence: PMID:25143406 In the construct GST-Pyk3-KI, where KI was expressed in isolation from KII, the TKL directed tyrosine phosphorylation of His-STATc on Y922. |
| GO:0004712 protein serine/threonine/tyrosine kinase activity | IEA GO_REF:0000003 | MARK AS OVER ANNOTATED | Summary: Derived from the UniProt EC 2.7.12.1 (dual-specificity kinase) assignment, which rests on the TKL family name rather than on any Ser/Thr phosphorylation data for Pyk3. Reason: Experimental data show tyrosine-directed activity (autophosphorylation on Tyr, STATc Tyr922). Recombinant Pyk3 had negligible activity towards the general Ser/Thr substrate myelin basic protein, and no Ser/Thr substrate is known. The tyrosine kinase term (GO:0004713) better reflects current evidence; dual specificity is not excluded but is unsupported. Supporting Evidence: PMID:25143406 Whereas Pyk2 directed efficient 32P incorporation into MBP, the Pyk3 activity was in comparison negligible |
| GO:0004713 protein tyrosine kinase activity | IDA PMID:24587195 Identification of the protein kinases Pyk3 and Phg2 as regul... | ACCEPT | Summary: Vu et al. 2014 show reduced STATc Tyr922 phosphorylation in pyk3- cells but, in the cached full text, do not assay Pyk3 kinase activity directly; the evidence is closer to IMP. The activity itself is well established by PMID:25143406. Reason: Protein tyrosine kinase activity is correct for Pyk3 and is its core activity; the evidence code from this particular paper is weak but the annotation should stand. Supporting Evidence: PMID:24587195 STATc Phosphorylation is Reduced in pyk3−, phg2− and pyk3−/phg2− Cells PMID:25143406 In the construct GST-Pyk3-KI, where KI was expressed in isolation from KII, the TKL directed tyrosine phosphorylation of His-STATc on Y922. |
| GO:0004713 protein tyrosine kinase activity | IDA PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | ACCEPT | Summary: Direct biochemical evidence - recombinant Pyk3 autophosphorylates on tyrosine (lost in K1084A kinase-dead mutant), the isolated KI domain phosphorylates STATc on Y922, and immunopurified myc-Pyk3 shows stress/cGMP-induced STATc tyrosine kinase activity. Reason: Best-supported core molecular function of Pyk3. Supporting Evidence: PMID:25143406 By analysis of cells expressing myc-tagged Pyk2 and Pyk3 in an in vitro kinase assay, we show that both enzymes directly increase tyrosine phosphorylation of STATc in response to cGMP. PMID:25143406 In the construct GST-Pyk3-KI, where KI was expressed in isolation from KII, the TKL directed tyrosine phosphorylation of His-STATc on Y922. |
| GO:0004713 protein tyrosine kinase activity | IDA PMID:8898113 Classification of tyrosine kinases from Dictyostelium discoi... | ACCEPT | Summary: Adler et al. 1996 identified DPYK3 by phosphotyrosine-antibody screening and showed tyrosine autophosphorylation of the bacterially expressed C-terminal fragment. Reason: Tyrosine autophosphorylation of recombinant protein is direct evidence for tyrosine kinase activity, confirmed by later work (PMID:25143406). Supporting Evidence: PMID:8898113 Two new kinases of Dictyostelium discoideum were identified by screening of a (lambda)gt11 expression library with a phosphotyrosine specific antibody. |
| GO:0004713 protein tyrosine kinase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Rhea-mapped electronic annotation matching the experimentally supported tyrosine kinase activity. Reason: Consistent with direct experimental evidence. Supporting Evidence: PMID:25143406 Tyrosine phosphorylation of Pyk3 was ablated when K1084, in the predicted site of ATP binding |
| GO:0005515 protein binding | IPI PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | MODIFY | Summary: Pull-down interaction between GST-Pyk3 and STATc (Q54BD4). The interaction is tyrosine-phosphorylation dependent and requires the conserved SH2-domain arginine (R831) of STATc, i.e. phosphorylated Pyk3 binds the STATc SH2 domain. Reason: Protein binding is uninformative. The interaction is specifically a phosphotyrosine-dependent docking of the STATc SH2 domain onto Pyk3, best captured by SH2 domain binding. Proposed replacements: SH2 domain binding Supporting Evidence: PMID:25143406 In addition, as with Pyk2, point mutation of the ultraconserved R residue (R831 equivalent to R175 in v-Src) within the STATc SH2 domain ablates binding to GST-Pyk3 |
| GO:0005524 ATP binding | IC PMID:8898113 Classification of tyrosine kinases from Dictyostelium discoi... | ACCEPT | Summary: Inferred from tyrosine kinase activity; ATP binding is a necessary feature of the catalytic TKL domain. Reason: Correct inference; the K1084A ATP-site mutant is kinase-dead. Supporting Evidence: PMID:25143406 Tyrosine phosphorylation of Pyk3 was ablated when K1084, in the predicted site of ATP binding |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO from kinase domain / ATP-binding site signatures; correct. Reason: Consistent with the conserved ATP-binding lysine of the catalytic domain. |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Cytoplasmic localization inferred for the TKL family node; consistent with Pyk3 being cytosolic and cortical. Reason: Pyk3 has no transmembrane segment and is experimentally cytosolic/cortical. Supporting Evidence: PMID:25143406 Before sorbitol treatment, Pyk3 is also enriched in the cytosol, but, after exposure to sorbitol, the amount at the cortex increases, where it often colocalizes with F-actin |
| GO:0005829 cytosol | IDA PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | ACCEPT | Summary: Immunofluorescence of myc-Pyk3 shows cytosolic enrichment in unstressed cells. Reason: Direct localization evidence. Supporting Evidence: PMID:25143406 Before sorbitol treatment, Pyk3 is also enriched in the cytosol, but, after exposure to sorbitol, the amount at the cortex increases, where it often colocalizes with F-actin |
| GO:0005938 cell cortex | IDA PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | ACCEPT | Summary: Stress-induced relocation of Pyk3 to the actin-rich cortex. Reason: Direct localization evidence; likely relevant to stress-specific signalling. Supporting Evidence: PMID:25143406 Before sorbitol treatment, Pyk3 is also enriched in the cytosol, but, after exposure to sorbitol, the amount at the cortex increases, where it often colocalizes with F-actin |
| GO:0006972 hyperosmotic response | IGI PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | ACCEPT | Summary: The pyk2-/pyk3- double mutant (P18161 = Pyk2/SplB) is completely unable to activate STATc in response to hyperosmotic sorbitol, and Pyk3 activity is stress-induced. Reason: Pyk3 is a stress-activated kinase that directly phosphorylates STATc in the hyperosmotic stress response; this is one of its core biological roles. Supporting Evidence: PMID:25143406 The strongest evidence for a functional interaction between Pyk2 and Pyk3 derives from the analysis of the stress reaction in the double null; it is entirely unresponsive. |
| GO:0007165 signal transduction | IBA GO_REF:0000033 | ACCEPT | Summary: General signalling-role inference for the TKL family; correct for Pyk3, which transduces stress signals to STATc. Reason: Correct though general; the specific role is captured by GO:1904894 and GO:0006972. Supporting Evidence: PMID:25143406 These data suggest that stress acts by stimulating Pyk2 and Pyk3 activity. |
| GO:0010628 positive regulation of gene expression | IGI PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | KEEP AS NON CORE | Summary: STATc-dependent genes are not induced by stress in the pyk2-/pyk3- double null. Reason: Correct but indirect downstream consequence of STATc activation; the core role is the STAT-activating kinase step. Supporting Evidence: PMID:25143406 The three genes are maximally induced in the parental (Ax2) strain and somewhat less well induced in the pyk2− or pyk3− strain |
| 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: pyk3- cells show diminished stress-induced transcription of STATc target genes. Reason: Valid but indirect consequence of reduced STATc activation. Supporting Evidence: PMID:24587195 Pyk3 and Phg2 are required for full transcriptional activation of STATc-dependent genes. |
| GO:0030587 sorocarp development | IGI PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | KEEP AS NON CORE | Summary: Double pyk2-/pyk3- disruption makes development highly stress sensitive. Reason: Developmental phenotype downstream of signalling; pleiotropic, not a core function. Supporting Evidence: PMID:25143406 However, the fact that simultaneous disruption of pyk2 and pyk3 renders development highly stress sensitive suggests that Pyk2 and Pyk3 have essential targets additional to STATc. |
| GO:0031156 regulation of sorocarp development | IMP PMID:18657170 Dictyostelium kinase DPYK3 negatively regulates STATc signal... | KEEP AS NON CORE | Summary: dpyk3- slugs show aberrant prestalk/prespore patterning (Lee et al. 2008). Araki et al. (PMID:25143406) could not reproduce the DIF-1 STATc phenotype in an Ax2 background, suggesting strain dependence. Reason: Supported developmental phenotype but downstream of the kinase's signalling role. Supporting Evidence: PMID:18657170 dpyk3- cells displayed aberrant pattern formation during development |
| GO:0042307 positive regulation of protein import into nucleus | IGI PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | KEEP AS NON CORE | Summary: Stress-induced nuclear enrichment of STATc is lost in the pyk2-/pyk3- double null; nuclear accumulation follows from STATc Tyr922 phosphorylation and dimerization. Reason: Indirect consequence of STATc phosphorylation; Pyk3 does not itself act on the nuclear import machinery. Supporting Evidence: PMID:25143406 (B) Nuclear enrichment of STATc in parental Ax2 and pyk2−, pyk3−, and double-null strains. PMID:24587195 Nuclear translocation of STATc is delayed in pyk3−, phg2−, and pyk3−/phg2− cells. |
| GO:0045595 regulation of cell differentiation | IMP PMID:18657170 Dictyostelium kinase DPYK3 negatively regulates STATc signal... | KEEP AS NON CORE | Summary: pstO zone not formed and prespore zone expanded in dpyk3- slugs. Reason: Cell-fate phenotype downstream of STATc signalling; not a core function. Supporting Evidence: PMID:18657170 The prestalk O zone was not properly formed and, instead, the prespore zone was expanded in dpyk3- slugs. |
| GO:0106310 protein serine kinase activity | IEA GO_REF:0000116 | REMOVE | Summary: Rhea mapping from the UniProt dual-specificity catalytic-activity lines (EC 2.7.12.1), which are not supported by experimental data for Pyk3. Reason: All biochemical data show tyrosine-directed activity; recombinant Pyk3 had negligible activity on the generic Ser/Thr substrate MBP, and no serine substrate of Pyk3 is known. Pyk3 was also shown not to be the kinase for the PTP3 serine sites. Supporting Evidence: PMID:25143406 Whereas Pyk2 directed efficient 32P incorporation into MBP, the Pyk3 activity was in comparison negligible PMID:24587195 These results exclude Pyk3 as a specific inhibitor of PTP3 |
| GO:1904894 positive regulation of receptor signaling pathway via STAT | IGI PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | ACCEPT | Summary: Pyk3 and Pyk2 redundantly drive STATc Tyr922 phosphorylation; the double null is non-activatable. Reason: Pyk3 is a JAK-analogous activator of STATc; this captures its core biological role. Supporting Evidence: PMID:25143406 Here we showed that the two most closely related of the five kinases, Pyk2 and Pyk3, are also functionally homologous, because they direct net phosphorylation of STATc when cells are subjected to hyperosmotic stress. |
| GO:1904894 positive regulation of receptor signaling pathway via STAT | IMP PMID:24587195 Identification of the protein kinases Pyk3 and Phg2 as regul... | ACCEPT | Summary: Reduced phospho-STATc, delayed STATc nuclear translocation and reduced target gene induction in pyk3- cells. Reason: Consistent with the direct STATc kinase activity of Pyk3. Note Lee et al. 2008 reported the opposite (negative) effect on DIF-1-induced STATc phosphorylation in a different strain background, not reproduced in Ax2. Supporting Evidence: PMID:24587195 STATc Phosphorylation is Reduced in pyk3−, phg2− and pyk3−/phg2− Cells |
| GO:0097696 cell surface receptor signaling pathway via STAT | IDA PMID:25143406 Two Dictyostelium tyrosine kinase-like kinases function in p... | NEW | Summary: Pyk3 directly phosphorylates the activating residue Tyr922 of STATc, a step of the STAT pathway itself, so it participates in the pathway rather than only regulating it. Reason: Matches the IDA annotation of the paralog Pyk2 (splB) for the same STATc Tyr922 kinase role. The kinase domain phosphorylates STATc Y922 in vitro, so Pyk3 performs a step of the pathway. It complements the existing GO:1904894 annotations and is neither an ancestor nor a descendant of any annotated term. Keeping both the pathway term and its positive-regulation term is deliberate and mirrors dictyBase practice for the paralog splB, which carries both in GOA. Supporting Evidence: PMID:25143406 In the construct GST-Pyk3-KI, where KI was expressed in isolation from KII, the TKL directed tyrosine phosphorylation of His-STATc on Y922. PMID:25143406 By analysis of cells expressing myc-tagged Pyk2 and Pyk3 in an in vitro kinase assay, we show that both enzymes directly increase tyrosine phosphorylation of STATc in response to cGMP. |
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Download this section (compressed HTML)Q: Which tyrosine residue(s) of Pyk3 are autophosphorylated and serve as the docking site for the STATc SH2 domain, and how does stress or cGMP (e.g. via GbpC) increase Pyk3 autophosphorylation?
Q: Does Pyk3 phosphorylate any serine/threonine substrates in vivo, justifying the dual-specificity (EC 2.7.12.1) name, or is it tyrosine-selective?
Q: Is the opposite STATc phenotype reported for dpyk3- in DIF-1 signalling (Lee et al. 2008) explained by strain background, and does Pyk3 have targets other than STATc during development?
Experiment: Phosphoamino-acid analysis and phosphoproteomics of autophosphorylated recombinant Pyk3 KI domain and of substrates in wild-type versus pyk3- (and pyk2-/pyk3-) stressed cells.
Hypothesis: Pyk3 is tyrosine-selective and lacks physiologically relevant Ser/Thr kinase activity.
Type: biochemical
Experiment: Map Pyk3 autophosphorylation sites by mass spectrometry, then test Tyr-to-Phe mutants for STATc SH2 binding in pull-downs and for rescue of STATc activation in pyk2-/pyk3- cells.
Hypothesis: Specific autophosphorylated tyrosines in Pyk3 recruit the STATc SH2 domain and are required for stress-induced STATc activation.
Type: mutagenesis
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