The defect in stalk formation which was observed in cell lines with strongly reduced CNB expression occurs due to smaller and incompletely vacuolated stalk cells and this result points to an important role for CNB or the CN holoenzyme in the regulation of stalk development in Dictyostelium. DIF-1 induces the expression of prestalk-specific genes via an increase in the intracellular free Ca2+ [12]. Based on our results we speculate that the effects of increased Ca2+i are mediated by CN. In analogy to the well-characterized role of CN in dephosphorylation of transcription factor complexes of the NFAT type we propose that calcineurin, in response to an increase in cytosolic Ca2+, dephosphorylates a cytosolic component of a transcription complex, which is then translocated to the nucleus and activates – in concert with nuclear components of the active complex – the transcription of prestalk genes. This hypothesis is directly supported by our observation that RNAi mutant 01–5 failed to properly induce expression of the prestalk-specific gene ecmB during development.
The phenotypic defects which we report here complement previous studies on the effects of mutations in the Dictyostelium glycogen synthase kinase-3 homolog GskA [18] which has been shown to promote posterior cell patterning and inhibit anterior cell differentiation [reviewed in [19]]. GskA is regulated through an intracellular protein tyrosine kinase/phosphatase pathway mediated by the cAMP receptors CAR1 and/or CAR3 and CAR4. GskA is activated by CAR1- and/or CAR3-mediated activation of the tyrosine kinase ZAK1 [20], and inactivated by CAR4-mediated activation of a protein tyrosine phosphatase [21]. GskA null cells show accelerated early development and form only stalk cells during morphogenesis [18]. Both the gskA null and the cnbA RNAi phenotypes are cell autonomous [18]; see above, fig. 8A. The direct link between GskA and CN is supported by recent results that come from analysis of the NFAT pathway. In T cells phosphorylation of NFAT by GSK3 represses NFAT-dependent gene expression by inhibition of NFAT binding to DNA [22]. Inhibition of re-export of NFAT to the cytosol when GSK3 is inactivated, leads to increased NFAT-dependent activation of gene expression [23].
Deletion of the Dictyostelium aarA gene, a homolog of mammalian β-catenin, leads to a closely similar breakdown of tip dominance and to formation of ectopic tips during late development as silencing of CNB. In both cases, the defect is non-cell autonomous [24]; see above, fig. 8B. It has been shown that AarA plays a necessary role in the formation of adherence junctions at the neck of the rising stalk [25]. β-catenin is also a transcriptional co-activator in the metazoan canonical Wnt pathway. In the control of dorsoventral axis formation in Xenopus by extracellular signals, a CN-independent NFAT mutant inhibited anterior development of the primary axis, whereas a dominant negative NFAT mutation induced ectopic dorsal axis formation and the expression of target genes of the canonical Wnt pathway, suggesting that CN and NFAT are part of the noncanonical Wnt/Ca2+ pathway which leads to inhibition of the canonical Wnt pathway upstream of β-catenin [26] (for a Wnt/Ca2+ pathway overview see [27]). It is therefore possible to propose a model for Dictyostelium tip dominance where a pathway similar to the canonical metazoan Wnt pathway is acting through CAR1/CAR3 to activate GskA, and a noncanonical "Wnt/Ca2+" pathway through CAR4 to activate CN via an intracellular Ca2+ signal. The postulated transcription factor target for CN in Dictyostelium is so far unknown.
Calcineurin B is a homolog of members of the 4 EF hand calcium sensor protein family which include, for example, recoverin, the neuronal calcium sensor-1 (NCS-1), and the plant calcineurin B-like proteins [(CBLs [28]]. It has recently been demonstrated that N myristoylation, a common feature of CNB and the Ca2+ sensor protein family, increases the cooperativity of Ca2+ binding to NCS-1 and leads to larger conformational changes upon Ca2+ binding than in the nonmyristoylated protein [29]. If Ca2+ sensing is indeed a major function of calcineurin B, the phenotypic defects observed in our cnbA RNAi mutants may be due to the failure to correctly sense the slow, sustained increase in Ca2+i from ca. 50 to ca. 150 nM during a period of about 8 hours [12], which was proposed to induce prestalk gene expression. Starting during early development D. discoideum cells express two CNB isoforms, one of them lacking the myristoylation consensus site. Further work has to show whether it is the myristoylated CNB isoform which plays a crucial role in the mediation of DIF effects on prestalk gene expression.
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