Two Dictyostelium tyrosine kinase-like kinases function in parallel, stress-induced STAT activation pathways.
Bottom Line: The site(s) that are generated bind the SH2 domain of STATc, and then STATc becomes the target of further kinase action.The latter functions, like the JH2 domain of metazoan JAKs, as a negative regulator of the kinase domain.The fact that two differently regulated kinases catalyze the same phosphorylation event may facilitate specific targeting because under stress, Pyk3 and Pyk2 accumulate in different parts of the cell; Pyk3 moves from the cytosol to the cortex, whereas Pyk2 accumulates in cytosolic granules that colocalize with PTP3.
Affiliation: College of Life Sciences, Welcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.Show MeSH
Mentions: Another major difference between Pyk2 and Pyk3 is the presence in Pyk3 of both a pseudokinase domain and an adjacent, C-terminus-proximal TKL domain. The metazoan JAKs have a similar structure, and there the pseudokinase domain of JAK2 serves to negatively regulate the kinase activity of the C-terminal tyrosine kinase domain. To determine whether Pyk3 uses a similar mechanism, we first established a system in which the kinase activity of recombinant GST-tagged enzyme could be assayed in-trans using isotopically γ-labeled ATP. The substrate chosen was myelin basic protein (MBP). Whereas Pyk2 directed efficient 32P incorporation into MBP, the Pyk3 activity was in comparison negligible (Figure 8A). We then concentrated on the TKL (KI) and pseudokinase (KII) domains of Pyk3 (Supplemental Figure S3), using histidine (His)-STATc as the substrate in an in vitro kinase reaction and monitoring the reaction using the phospho-STATc antibody CP22 (Figure 8B). In the construct GST-Pyk3-KI, where KI was expressed in isolation from KII, the TKL directed tyrosine phosphorylation of His-STATc on Y922. In contrast, in the construct GST-Pyk3-KII+KI, where KI was expressed in the presence of KII, the TKL domain did not direct tyrosine phosphorylation of His-STATc on Y922. Thus the presence of the KII domain represses the kinase activity of the KI domain.
Affiliation: College of Life Sciences, Welcome Trust Biocentre, University of Dundee, Dundee DD1 5EH, United Kingdom.