Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the C␣ or RI␣ subunit gene of PKA in an expression vector, which upregulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RII␤ subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the C␣ gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH 2-terminal myristyl group of C␣ is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.

Activating mutations in codon D816 of the tyrosine kinase receptor, KIT, are found in the majority of patients with systemic mastocytosis. We found that the transcription factor, microphthalmiaassociated transcription factor (MITF), is highly expressed in bone marrow biopsies from 9 of 10 patients with systemic mastocytosis and activating c-KIT

The Microphthalmia-associated transcription factor (Mitf) is an essential basic helix-loop-helix leucine zipper transcription factor for mast cell development. Mice deficient in Mitf harbor a severe mast cell deficiency, and Mitf-mutant mast cells cultured ex vivo display a number of functional defects. Therefore, an understanding of the genetic program regulated by Mitf may provide important insights into mast cell differentiation. Multiple, distinct isoforms of Mitf have been identified in a variety of cell types; we found that Mitf-a, Mitf-e, and Mitf-mc were the major isoforms expressed in mast cells. To determine the physiologic function of Mitf in mast cells, we restored expression of these isoforms in primary mast cells from Mitf ؊/؊ mice. We found that these isoforms restored granular morphology and integrin-mediated migration. By microarray analysis, proteases, signaling molecules, cell surface receptor, and transporters comprised the largest groups of genes up-regulated by all isoforms. Furthermore, we found that isoforms also regulated distinct genes sets, suggesting separable biological activities. This work defines the transcriptome regulated by Mitf in mast cells and supports its role as master regulator of mast cell differentiation. Expression of multiple isoforms of this transcription factor may provide for redundancy of biological activities while also allowing diversity of function. (2), congestive heart failure (3), and multiple sclerosis (4). Although mast cells have the capacity to promote diverse pathologic states, they also participate in normal physiologic processes, such as innate immunity against parasitic and bacterial infections (5, 6). These diverse functions may be regulated by distinct and shared pathways. A dissection of these pathways may allow selective regulation of these processes for therapeutic benefit.A number of transcription factors play essential roles in mast cell development, including the zinc finger factors GATA-1 (7) and GATA-2 (8), the Ets family member PU.1 (9), and the helixloop-helix leucine zipper factor, Microphthalmia-associated transcription factor (Mitf) 4 (10, 11 To date, nine distinct isoforms of Mitf have been identified (22). These isoforms arise from an alternative splicing event that joins a unique first exon with the common body of the gene. The resulting proteins possess distinct amino termini, but share transactivation, DNA binding, and dimerization motifs. The expression of alternatively spliced transcripts may be controlled by distinct promoter/ enhancer elements, allowing for differential regulation of isoforms. Furthermore, transcripts that encode for unique proteins species may possess distinct biological functions. Alternative splicing of mRNA transcripts is a widespread mechanism for generating diversity in multicellular organisms. Thirty-five to 65% of human genes are predicted to undergo alternative splicing, the majority of which encode for distinct protein species (23). This mechanism is particularly widespread in complex systems such ...

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