RGC-32 Increases p34CDC2 Kinase Activity and Entry of Aortic Smooth Muscle Cells into S-phase

Abstract

Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34 CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34 CDC2 -cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34 CDC2 -mediated phosphorylation and resulted in loss of p34 CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34 CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34 CDC2 .C5b-9, the membrane attack complex of complement, causes cell death by forming transmembrane pores (1). When the number of C5b-9 molecules is limited to a sublytic level, nucleated cells are able to escape cell death by eliminating membrane-inserted terminal complement complexes (TCC 1 ; C5b-7, C5b-8, and C5b-9) by endocytosis and/or membrane shedding (2-4). Among these complexes, C5b-9 is most potent in activating target cells. C5b-9 causes a Ca 2ϩ influx and generates intracellular second messengers, including phosphatidylinositol triphosphates, diacylglycerol, and ceramide (5-8). Membrane-inserted TCC activates the G i /G o family of G proteins (9). Activation of G i /G o by TCC is responsible for the G␤␥-mediated activation of cell cycle through activation of Ras, Raf-1, extracellular signal regulated kinase-1 (10), and activation of phosphatidylinositol 3-phosphate kinase (10, 11).Cell cycle activation by C5b-9 is associated with an increase in CDK4, CDK2, and p34 CDC2 activities, and this is followed by an increase in DNA synthesis and cell proliferation (11, 12, 24 -26). The C5b-9-induced DNA synthesis is abolished by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1 and phosphatidylinositol 3-phosphate kinase (11). Cell cycle activation by C5b-9 in postmitotic cells, such as oligodendrocytes (OLG) and myotubes, is associated with expression of c-JUN and c-FOS protooncogenes and loss of differentiation (12)(13)(14).In an effort to find novel C5b-9-induced genes involved in cell cycle regulation, we cloned the rat Response Gene to Complement (RGC-32) using mRNA differential display PCR in OLG (15, 16). C5b-9 en...

Response Gene to Complement 32, a Novel Regulator for Transforming Growth Factor-β-induced Smooth Muscle Differentiation of Neural Crest Cells

“…RGC-32 was first identified in oligodendrocytes in response to complement activation (38). It is a 14-kDa protein expressed in many adult human tissues including heart, brain, liver, skeletal muscle, placenta, kidney, and pancreas (39), and it is overexpressed in colon cancer and many tumors (40). Functionally, RGC-32 has been shown to play a role in cell cycle activation.…”

RGC32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in G2/M arrest

“…Overexpression of RGC32 induces quiescent aortic smooth-muscle cells to enter into the S-phase (Badea et al, 2002). Others have reported that RGC32 physically associated with cyclin-dependent kinase p34 CDC2 , and that its kinase activity increased through phosphorylation by p34 CDC2 -cyclin B1 (Badea et al, 2002).…”

Cancer and the Complement Cascade

“…In response to the MAC, RGC-32 protein shows complex formation with cyclin B1/CDC2, stimulating the cell cycle in rat oligodendrocytes (58) and human aortic smooth muscle cells (59). Upregulation of RGC-32 mRNA and protein is an especially noteworthy effect given the widespread presence of RGC-32 in cancer.…”