No abstract

SUMMARYIn Dictyostelium, the interaction of secreted cAMP with specific cell surface receptors regulates the activation/de-activation of GSK3, which mediates developmental cell patterning. In addition, Dictyostelium cells polarize in response to extracellular cAMP, although a potential role for GSK3 in this pathway has not been investigated. Previously, we had shown that ZAK1 was an activating tyrosine kinase for GSK3 function in Dictyostelium and we now identify ZAK2 as the other tyrosine kinase in the cAMPactivation pathway for GSK3; no additional family members exist. We also now show that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 separately regulate GSK3 in distinct differentiated cell populations, and that ZAK2 acts in both autonomous and non-autonomous pathways to regulate these cell-type differentiations. Finally, we demonstrate that efficient polarization of Dictyostelium towards cAMP depends on ZAK1-mediated tyrosine phosphorylation of GSK3. Combinatorial regulation of GSK3 by ZAK kinases in Dictyostelium guides cell polarity, directional cell migration and cell differentiation, pathways that extend the complexity of GSK3 signaling throughout the development of Dictyostelium.

DPYK3, a member of the Dictyostelium TKL (tyrosine kinase like) kinase family, was ablated by homologous recombination. dpyk3 − cells displayed aberrant pattern formation during development. The prestalk O zone was not properly formed and, instead, the prespore zone was expanded in dpyk3 − slugs. During development, the transcription factor STATc (signal transducers and activators of transcription c) was persistently phosphorylated and ecmAO expression level was kept low in dpyk3 − cells. Furthermore, in response to differentiation inducing factor-1 (DIF-1) in suspension culture, dpyk3 − cells displayed persistent STATc phosphorylation and reintroduction of DPYK3 in dpyk3 − cells restored transient STATc phosphorylation similarly to wild type cells. In contrast to the positive STAT regulation by Janus Kinase in metazoans, Dictyostelium DPYK3 negatively regulates STATc during development in response to DIF-1 signaling.

The novel Dictyostelium phosphatase MPL1 contains six leucine-rich repeats at the amino-terminal end and a phosphatase domain at the carboxyl end. Similarly architectured phosphatases exist among other protozoa, such as Entamoeba histolytica, Leishmania major, and Trypanosoma cruzi. MPL1 was strongly induced after 5 h of development; ablation by homologous recombination led to defective streaming and aggregation during development. In addition, cyclic AMP (cAMP)-pulsed mpl1 ؊ cells showed reduced random and directional motility. At the molecular level, mpl1؊ cells displayed higher prestimulus and persistent poststimulus ERK2 phosphorylation in response to cAMP stimulation. Consistent with their phenotype of persistent ERK2 phosphorylation, mpl1؊ cells also displayed an aberrant pattern of cAMP production, resembling that of the regA ؊ cells. Reintroduction of a full-length MPL1 into mpl1 ؊ cells restored aggregation, ERK2 regulation, random and directional motility, and cAMP production similar to wild-type cells. We propose that MPL1 is a novel phosphatase essential for proper regulation of ERK2 phosphorylation and optimal motility during development.Mitogen-activated protein kinases (MAPKs) are central in the regulation of proliferation, differentiation, and cell migration in diverse eukaryotic cells (10,13,21,25,26,27). The MAP kinase ERK2 also plays critical roles during Dictyostelium development. ERK2 is essential for initiation and propagation of periodic cyclic AMP (cAMP) pulses during aggregation and differentiation. Chemoattractants, such as cAMP, induce ERK2 activation. Activated ERK2 subsequently inhibits the intracellular cAMP-specific phosphodiesterase RegA, resulting in an increase in the cytosolic cAMP level (15,19,22). erk2 Ϫ cells, starved for 8 h, exhibited a decrease in motility and a severe chemotaxis defect toward a cAMP gradient. Aberrancy in chemotaxis was aggravated in the presence of a strong cAMP gradient (2 M) compared to a weak one (0.1 M) (27). erk2 Ϫ cells also display defective cytoskeletal remodeling in response to chemoattractant stimulation. A polarized wild-type cell typically displays a single dominant leading edge enriched with F-actin. Myosin II localizes to the lateral side and back of a polarized cell, where it functions to suppress lateral pseudopods and provides tractional force to the back. This single dominant leading edge disintegrates but forms again after 7 min in response to global cAMP stimulation (27). In contrast, erk2 Ϫ cells, under the same condition, displayed multiple crown-like membranous protrusions, which were enriched not only in F-actin but also in myosin II (27). This aberrant structure, which was proposed to be less stable and unable to provide necessary traction force for cells to move, is believed to be the reason why erk2 Ϫ cells are less motile than wild-type cells (27).It is well-established that the dual phosphorylation of threonine 176 and tyrosine 178 residues of the ERK2 activation loop, often called the TEY motif, mediates activation of ERK2...