The regulation of alternative mRNA splicing factors by extracellular cues and signal transduction cascades is poorly understood. Using an engineered extracellular signal-regulated kinase 2 (ERK2) that can utilize ATP analogs, we have identified the alternative mRNA splicing factor 45 (SPF45), which is overexpressed in cancer, as a novel coimmunoprecipitating ERK2 substrate. ERK2 phosphorylated SPF45 on Thr71 and Ser222 in vitro and in cells in response to H-RasV12, B-RAF-V600E, and activated MEK1. Jun N-terminal kinase 1 (JNK1) and p38␣ also phosphorylated SPF45 in vitro and associated with SPF45 in cells. SPF45 was differentially phosphorylated in cells by all three mitogen-activated protein (MAP) kinases in response to phorbol myristate acid (PMA), H 2 O 2 , UV, and anisomycin stimulation. ERK and p38 activation decreased SPF45-dependent exon 6 exclusion from fas mRNA in a minigene assay in cells. Stable overexpression of SPF45 in SKOV-3 cells dramatically inhibited cell proliferation in a phosphorylation-dependent manner through inhibition of ErbB2 expression. SPF45 overexpression also induced EDA inclusion into fibronectin transcripts and fibronectin expression in a phosphorylation-dependent and -independent manner, respectively, specifically affecting cellular adhesion to a fibronectin matrix. These data identify SPF45 as the first splicing factor regulated by multiple MAP kinase pathways and show effects of both SPF45 overexpression and phosphorylation.T he expression of more than one protein from a single gene is regulated by alternative mRNA splicing, in which the exons from pre-mRNA of a transcribed gene are differentially spliced together (6), affecting the composition of the final protein product. Alternative splicing is thought to regulate between 60 and 74% of the human genome (42, 66), and up to 50% of human genetic diseases arise from changes in alternative splicing (38). Pre-mRNA splicing is regulated by both small nuclear ribonucleoprotein particles (snRNPs) and proteins that function in the stepwise processing of pre-mRNA (29, 65). Alternative splicing is primarily regulated by the hnRNP (heterogenous nuclear ribonucleoproteins) and SR (serine-arginine-rich) families of splicing factor proteins (28,37,41). Other alternative splicing factors fall outside these families and contain one or more RNA recognition motifs (RRMs) and protein-protein binding domains. Splice site selection depends on the relative concentrations of these proteins (8,27) and is regulated by reversible phosphorylation (57). Little is known about how extracellular signals and intracellular signal transduction regulate pre-mRNA splicing.The alternative mRNA splicing factor SPF45 (splicing factor 45) was identified in mass spectrometry analysis of the spliceosome complex (45) and acts in the second step of splicing, regulating 3= recognition of alternative splice sites in pre-mRNA of the sxl gene in Drosophila (33). SPF45 regulates alternative splicing of pre-mRNA encoding the death receptor fas in minigene assays in cells, ind...

Alternative mRNA splicing is a mechanism to regulate protein isoform expression and is regulated by alternative splicing factors. The alternative splicing factor 45 (SPF45) is overexpressed in cancer, although few biological effects of SPF45 are known, and few splicing targets have been identified. We previously showed that Extracellular Regulated Kinase 2 (ERK2) phosphorylation of SPF45 regulates cell proliferation and adhesion to fibronectin. In this work, we show that Cdc2-like kinase 1 (Clk1) phosphorylates SPF45 on eight serine residues. Clk1 expression enhanced, whereas Clk1 inhibition reduced, SPF45-induced exon 6 exclusion from Fas mRNA. Mutational analysis of the Clk1 phosphorylation sites on SPF45 showed both positive and negative regulation of splicing, with a net effect of inhibiting SPF45-induced exon 6 exclusion, correlating with reduced Fas mRNA binding. However, Clk1 enhanced SPF45 protein expression, but not mRNA expression, whereas inhibition of Clk1 increased SPF45 degradation through a proteasome-dependent pathway. Overexpression of SPF45 or a phospho-mimetic mutant, but not a phospho-inhibitory mutant, stimulated ovarian cancer cell migration and invasion, correlating with increased fibronectin expression, ERK activation and enhanced splicing and phosphorylation of full-length cortactin. Our results demonstrate for the first time that SPF45 overexpression enhances cell migration and invasion, dependent on biochemical regulation by Clk1.

On the basis of our analysis of the SEER database, sublobar resection can be performed in selected patients with small tumors (≤2 cm) and early-stage disease (stage I/II). Although perioperative mortality is significant, the favorable 1-year and 3-year survival may justify the role of an additional procedure on the single lung.

Spray cryotherapy was a safe adjunct modality to accomplish airway patency in patients with benign tracheal stenosis. Although efficacy evidence is limited for SCT, it may be useful for patients who have experienced treatment failure with conventional modalities. Further analysis of this cohort will determine the physiologic durability of the reported short-term changes. Additional trials are warranted for further evaluation of this modality.

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