Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine͞threonine phosphatases in STAT3 signaling in human antigen-specific CD4 ؉ T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1͞PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine͞threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine͞threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogenactivated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.STATs (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that upon activation translocate into the nucleus where they activate target genes (reviewed in ref. 1). At present, seven STATs have been cloned, all of which have an Src homology 2 domain near their carboxyl terminus and a tyrosine residue near position 700 (e.g., Y705 in STAT3). Upon ligation, cytokine and growth factor receptor-associated Janus kinases (JAKs) become activated, possibly by transphosphorylation and͞or autophosphorylation. Once activated, JAKs phosphorylate the receptor on key tyrosine residues, which leads to recruitment of STAT proteins, which in turn are tyrosine-phosphorylated by JAKs. Phosphorylated STAT proteins homodimerize or heterodimerize through reciprocal Src homology 2-phosphotyrosine interactions and translocate to the nucleus where they bind specific DNA elements and regulate transcriptional activity of target genes (reviewed in refs. 1-3).STATs also are serine-phosphorylated in response to ligation of many cytokine and growth factor receptors (reviewed in ref. 4). The major site for serine phosphorylation in STAT1 and STAT3 is residue 727 (5), allthough additional serine phosphorylation sites have been proposed (6). Serine phosphorylation of STAT proteins modulate the DNA binding and͞or transcri...

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Centrin, an EF hand Ca 2ϩ binding protein, has been cloned in Tetrahymena thermophila. It is a 167 amino acid protein of 19.4 kDa with a unique N-terminal region, coded by a single gene containing an 85-base pair intron. It has Ͼ 80% homology to other centrins and high homology to Tetrahymena EF hand proteins calmodulin, TCBP23, and TCBP25. Specific cellular localizations of the closely related Tetrahymena EF hand proteins are different from centrin. Centrin is localized to basal bodies, cortical fibers in oral apparatus and ciliary rootlets, the apical filament ring and to inner arm (14S) dynein (IAD) along the ciliary axoneme. The function of centrin in Ca 2ϩ control of IAD activity was explored using in vitro microtubule (MT) motility assays. Ca 2ϩ or the Ca 2ϩ -mimicking peptide CALP1, which binds EF hand proteins in the absence of Ca 2ϩ , increased MT sliding velocity. Antibodies to centrin abrogated this increase. This is the first demonstration of a specific centrin function associated with axonemal dynein. It suggests that centrin is a key regulatory protein for Tetrahymena axonemal Ca 2ϩ responses, including ciliary reversal or chemotaxis.

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