A human fibroblast cDNA expression library was screened for cDNA clones giving rise to f lat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.Mutations of ras protooncogenes are found in a large variety of human tumors (1). It has been well established that Ras proteins are essential components in various intracellular signaling pathways involved in regulating gene expression and several other aspects of cellular behavior (2). Therefore, it is now important to find targets for these signals relevant to the expression of the malignant phenotype to understand the mechanism of cell transformation and to develop means to cure or prevent cancers.To this end, we have been isolating and characterizing genes that induce flat morphology (or ''flat reversion'') when expressed in a v-Ki-ras-transformed NIH 3T3 cell line, DT (3). The Krev-1 gene (4), also known as rap1A, which encodes a Ras-related protein containing a region identical to the effector domain of Ras, was isolated in a previous study (5) by using a plasmid-based human fibroblast cDNA expression library. Using a similar approach, Cutler et al. (6) isolated another transformation suppressor gene, rsp-1, encoding a leucinerich-repeat protein. Recently, we performed a similar screen of a human fibroblast cDNA expression library constructed with a new phagemid shuttle vector and obtained two cDNA clones exhibiting significant biological activities. One of these, clone CT124, was found to encode a truncated form of the MSX-2 homeobox protein, which induces flat reversion through a dominant-negative mechanism over the endogenous MSX-2 protein (7).Here we describe some properties of the other reversioninducing gene named RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) and its product. This reversioninducing gene is unique in that it encodes an extracellular protein with protease inhibitor-like domains and its expression is suppressed strongly in many tumors and cells transformed by various kinds of oncogenes. Restored expression of the RECK gene inhibits the invasive and metastatic activities of tumor cells. We also found that RECK negatively regulates matrix metalloproteinase-9 (MMP-9) (8) in two ways: suppression of MMP-9 secretion from the cells and direct inhibition of its enzymatic activity. T...

During humoral immune responses, two distinct genetic modification events diversify the Ig genes in germinal center (GC) B cells: somatic hypermutation and class switch recombination (CSR). Both processes require the activity of activation-induced cytidine deaminase (AID), an enzyme expressed specifically in GC B cells. However, the mechanisms that regulate AID activity are largely unknown. Here we report that protein kinase A (PKA) phosphorylates AID and regulates its activity in GC B cells. AID physically interacts with the PKA holoenzyme in the cytoplasm and is phosphorylated by the PKA catalytic subunit at specific residues. AID phosphorylation is required for CSR, because substitution of the two phosphorylation targets impairs its ability to rescue CSR in AID-deficient B cells. Pharmacologic inhibition of PKA prevents isotype class switching in a murine B-cell lymphoma cell line; conversely, B cells from mice where PKA activity is made constitutive by conditional deletion of the PKA regulatory subunit gene display enhanced CSR. These findings implicate PKA in the regulation of AID function and suggest that the control of T cell-dependent immune responses may be modulated, via AID, by signals that activate PKA.class switch recombination ͉ phosphorylation D uring T cell-dependent immune responses, the antibody genes are diversified in germinal center B cells by two orderly regulated DNA modification mechanisms. Somatic hypermutation (SHM) introduces single-nucleotide substitutions into the variable region of the Ig genes, allowing the selection of B cells that produce high-affinity antibodies (1, 2). Class switch recombination (CSR) is an intrachromosomal deletion͞recombination event that replaces the heavy-chain constant region (C H ) from C to downstream C H segments, thereby conferring distinct effector functions to the antibodies without affecting their specificity for the antigen (3, 4).Both SHM and CSR depend on activation-induced cytidine deaminase (AID), a B cell-specific deaminase whose expression is restricted to germinal center B cells and activated B cells (5-8). However, the mechanism by which AID initiates these DNA modification events remains a matter of debate. Although initial studies suggested that AID functions as an RNA-editing enzyme (8-10), substantial evidence indicates that AID can exert cytidine deaminase activity directly on DNA (11-16). Analysis of loss-offunction AID mutants suggests that interaction with specific cofactors is required to differentially regulate its activity on such independent processes: SHM and CSR (17,18). Posttranslational modifications of AID have also been evoked as an essential requirement for its deaminase activity. Indeed, it was recently shown that AID interacts specifically with replication protein A in a manner that is phosphorylation-dependent when targeting double-stranded DNA on in vitro-transcribed SHM substrates (19). The identification of signals regulating AID is critical to understand how SHM and CSR are controlled and will also help in elucidat...

B cell receptor (BCR) signaling plays a critical role in B cell tolerance and activation. Here, we show that mice with B cell-specific ablation of both Cbl and Cbl-b (Cbl-/-Cblb-/-) manifested systemic lupus erythematosus (SLE)-like autoimmune disease. The Cbl double deficiency resulted in a substantial increase in marginal zone (MZ) and B1 B cells. The mutant B cells were not hyperresponsive in terms of proliferation and antibody production upon BCR stimulation; however, B cell anergy to protein antigen appeared to be impaired. Concomitantly, BCR-proximal signaling, including tyrosine phosphorylation of Syk tyrosine kinase, Phospholipase C-gamma2 (PLC-gamma2), and Rho-family GTP-GDP exchange factor Vav, and Ca2+ mobilization were enhanced, whereas tyrosine phosphorylation of adaptor protein BLNK was substantially attenuated in the mutant B cells. These results suggested that the loss of coordination between these pathways was responsible for the impaired B cell tolerance induction. Thus, Cbl proteins control B cell-intrinsic checkpoint of immune tolerance, possibly through coordinating multiple BCR-proximal signaling pathways during anergy induction.

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