Cancer cells undergo epithelial-mesenchymal transition (EMT) as a program of increased invasion and metastasis during cancer progression. Here, we report that a novel regulator of EMT in cancer cells is protein kinase D1 (PKD1), which is downregulated in advanced prostate, breast, and gastric cancers. Ectopic reexpression of PKD1 in metastatic prostate cancer cells reversibly suppressed expression of mesenchyme-specific genes and increased epithelial markers such as E-cadherin, whereas small interfering RNA-mediated knockdown of PKD1 increased expression of mesenchyme markers. Further, PKD1 inhibited tumor growth and metastasis in a tumor xenograft model. PKD1 phosphorylates Ser 11 (S11) on transcription factor Snail, a master EMT regulator and repressor of E-cadherin expression, triggering nuclear export of Snail via 14-3-3σ binding. Snail S11 mutation causes acquisition of mesenchymal traits and expression of stem cell markers. Together, our results suggest that PKD1 functions as a tumor and metastasis suppressor, at least partly by regulating Snail-mediated EMT, and that loss of PKD1 may contribute to acquisition of an aggressive malignant phenotype.

Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. We show that mutations in the Drosophila LAMMER kinase, Doa, alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated similarly to dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing.

Clk/STY, the murine homologue of the recently described LAMMER family of protein kinases, autophosphorylates on serine/threonine and tyrosine residues in vitro and in vivo. LAMMER kinases are found throughout eukaryotes and possess virtually complete amino acid identity in many domains critical for kinase function, leading to the question of whether other family members also possess dual specificity. We report here that the Drosophila family member DOA, human SK-G1, and the Saccharomyces cerevisiae KNS1, all possess protein kinase activity and autophosphorylate with dual specificity in vitro, suggesting that the entire family possesses this activity. Although the LAMMER kinases are closely related to the mitogen-activated protein kinase family, they possess different substrate specificity in vitro, based on phosphorylation of peptide and protein substrates and sequencing of a phosphorylation site in a common substrate.Protein kinases are generally differentiated according to the specificity with which they phosphorylate substrates, with activity toward serine/threonine or tyrosine residues (1, 2). Recently, some kinases capable of phosphorylating all three amino acid residues have been identified (reviewed in Refs. 3 and 4). Such "dual specificity" kinases would be classified as serine/threonine-specific based on their amino acid sequences. Among these is murine STY or Clk (5, 6), whose cDNAs were isolated because their products autophosphorylated on tyrosine residues. Data base searches with the sequence of cDNAs from the Doa locus of Drosophila revealed murine Clk and other closely related homologues in eukaryotes ranging from yeast to humans (7). Additional homologues have subsequently been described in green plants (8) 1 (also accession number D49304 from the rice genome sequencing project), from rat (accession number X94351), and from Schizosaccharomyces pombe (accession number Z69239), a third family member in humans (9), a mink family member whose message is induced by cycloheximide in cultured cells (10), and a member from S. pombe, found in the genome sequencing project of this organism (accession number Z69239). We have dubbed these the LAMMER protein kinases, based upon the existence of this motif, or conserved variations upon it, in all members (7).The LAMMER kinases are nearly identical in size, spacing, and placement of their kinase catalytic domains and also show extremely high amino acid identity in domains essential for phosphotransfer to the peptide substrate and in substrate recognition (11). By analogy with the structure of two crystallized protein kinases (12, 13), the LAMMER motif lies in an ␣-helix below the substrate-binding cleft, potentially allowing it to make contact with substrates, and suggesting that these molecules are also highly conserved. Much of what is known of LAMMER kinase function derives from analysis of the Drosophila locus Doa, whose mutants were isolated during screens for loci with transcriptional regulatory effects (14,15). Doa is an essential gene; its mutations alt...

In recent years, the use of natural products for cancer prevention and treatment has received considerable attention. Bryostatin 1 is a natural macrocyclic lactone and a protein kinase D (PKD) modulator with potent antineoplastic properties that has been used to treat human cancers in clinical trials with limited success. Further understanding the mechanistic basis of Bryostatin 1 action may provide opportunities to improve clinical results of treatment with Bryostatin 1. We identified that PKD1, founding member of PKD family of serine/threonine kinases, modulates E-cadherin/B-catenin activity, which plays an important role in cell integrity, polarity, growth, and morphogenesis. An aberrant expression and localization of E-cadherin/B-catenin has been strongly associated with cancer progression and metastasis. In this study, we examined the effect of Bryostatin 1 treatment on PKD1 activation, B-catenin translocation and transcription activity, and malignant phenotype of prostate cancer cells. Initial activation of PKD1 with Bryostatin 1 leads to colocalization of the cytoplasmic pool of B-catenin with PKD1, trans-Golgi network markers, and proteins involved in vesicular trafficking. Activation of PKD1 by Bryostatin 1 decreases nuclear B-catenin expression and B-catenin/TCF transcription activity. Activation of PKD1 alters cellular aggregation and proliferation in prostate cancer cells associated with subcellular redistribution of E-cadherin and B-catenin. For the first time, we have identified that Bryostatin 1 modulates B-catenin signaling through PKD1, which identifies a novel mechanism to improve efficacy of Bryostatin 1 in clinical settings. [Mol Cancer Ther 2008;7(9):2703 -12]

B-Catenin is essential for E-cadherin-mediated cell adhesion in epithelial cells and also acts as a key cofactor for transcription activity. We previously showed that protein kinase D1 (PKD1), founding member of the PKD family of signal transduction proteins, is down-regulated in advanced prostate cancer and interacts with E-cadherin. This study provides evidence that PKD1 interacts with and phosphorylates B-catenin at Thr 112 and Thr 120 residues in vitro and in vivo; mutation of Thr 112 and Thr 120 results in increased nuclear localization of B-catenin and is associated with altered B-catenin-mediated transcription activity. It is known that mutation of Thr 120 residue abolishes binding of B-catenin to A-catenin, which links to cytoskeleton, suggesting that PKD1 phosphorylation of Thr 120 could be critical for cell-cell adhesion. Overexpression of PKD1 represses B-catenin-mediated transcriptional activity and cell proliferation. Epistatic studies suggest that PKD1 and E-cadherin are within the same signaling pathway. Understanding the molecular basis of PKD1-B-catenin interaction provides a novel strategy to target B-catenin function in cells including prostate cancer.