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In order to evaluate links between Ca2+/calmodulin (CaM)-dependent protein kinase type II (CaMK-II) and cell cycle progression, CaMK-II binding partners were sought in proliferating cells by epitope-tag tandem mass spectrometry. One protein identified was the gelsolin family member, flightless-I (Fli-I). Fli-I is not a CaMK-II substrate, but binds directly and preferentially to constitutively active (T287D) CaMK-II over inactive CaMK-II. Fli-I gradually enters the nucleus upon CaMK-II inhibition and is retained in the cytosol by T287D CaMK-II. CaMK-II inhibition and Fli-I overexpression suppress transcription of β-catenin dependent transcriptional reporters, whereas Fli-I suppression enhances their transcription. These findings support a novel mechanism whereby cytosolic CaMK-II influences β-catenin dependent gene expression through Fli-I. Structured summary MINT- 6603828 : delta CaMK-II (uniprotkb: Q6PHZ2 ) binds (MI: 0407 ) to Flightless-I (uniprotkb: Q9JJ28 ) by anti bait coimmunoprecipitation (MI: 0006 ) MINT- 6603847 : delta CaMK-II (uniprotkb: Q6PHZ2 ) phosphorylates (MI: 0217 ) MAP2 (uniprotkb: P20357 ) by protein kinase assay (MI: 0424 ) MINT- 6603768 : beta CaMK-II (uniprotkb: P28652 ) physically interacts (MI: 0218 ) with Flightless-I (uniprotkb: Q9JJ28 ) by anti bait coimmunoprecipitation (MI: 0006 ) MINT- 6603759 , MINT- 6603776 , MINT- 6603786 : delta CaMK-II (uniprotkb: Q6PHZ2 ) physically interacts (MI: 0218 ) with Flightless-I (uniprotkb: Q9JJ28 ) by anti bait coimmunoprecipitation (MI: 0006 ) MINT- 6603724 : CaMK-II delta (uniprotkb: Q6PHZ2 ) physically interacts (MI: 0218 ) with beta 5tubulin (uniprotkb: P99024 ), beta 2c tubulin (uniprotkb: P68372 ), alpha 1a tubulin (uniprotkb: P68369 ), b-Actin (uniprotkb: P60710 ), Tropomodulin-3 (uniprotkb: Q9JHJ0 ), Flightless-I (uniprotkb: Q9JJ28 ), FLAP1 (uniprotkb: Q3UZ39 ) and FLAP2 (uniprotkb: Q91WK0 ) by anti tag coimmunoprecipitation (MI: 0007 ).

Activation of α 1B adrenergic receptors (α 1B AR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of α 1B AR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express α 1B AR, were stably transfected with a rat α 1B AR cDNA (TFG2 cells), in order to study the effects of maintained α 1B AR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [ 3 H]thymidine incorporation into DNA. Stimulation of α 1B AR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G 0 /G 1 and G 2 /M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42 MAP kinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21 Cip1/WAF1 . Treatment of TFG2 cells with the specific MEK1 inhibitor PD98059, or infection with a −/− MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [ 3 H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 −/− blunted the ability of phenylephrine to increase p21 Cip1/WAF1 expression. In agreement with a role for increased p21 Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21 Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21 Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21 Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of α 1B ARs in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via α 1B AR appears to be p42 MAP kinase and p21 Cip1/WAF1 dependent.