TIMAP, a novel CAAX box protein regulated by TGF-β1 and expressed in endothelial cells

Abstract

Representational difference analysis of the glomerular endothelial cell response to transforming growth factor-beta1 (TGF-beta1) revealed a novel gene, TIMAP (TGF-beta-inhibited membrane-associated protein), which contains 10 exons and maps to human chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed in all cultured endothelial and hematopoietic cells. The frequency of the TIMAP SAGE tag is much greater in endothelial cell SAGE databases than in nonendothelial cells. Immunofluorescence studies of rat tissues show that anti-TIMAP antibodies localize to vascular endothelium. TGF-beta1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process. The TIMAP protein contains five ankyrin repeats, a protein phosphatase-1 (PP1)-interacting domain, a COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3, and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein localized to the plasma membrane in a CAAX box-dependent fashion. Hence, TIMAP is a novel gene highly expressed in endothelial and hematopoietic cells and regulated by TGF-beta1. On the basis of its domain structure, TIMAP may serve a signaling function, potentially through interaction with PP1.

Phosphorylation of Myosin Phosphatase Targeting Subunit 3 (MYPT3) and Regulation of Protein Phosphatase 1 by Protein Kinase A

“…They have an N-terminal PP1c-binding RVXF motif (KQVLF in MYPT3 and KVSF in TIMAP), and ankyrin repeats are homologous with all other family members, but their C terminus is markedly divergent. The C-terminal leucine zipper motif, a domain involved in many proteinprotein interactions and dimerization in MYPT1 (7,31), is not present in MYPT3 and TIMAP, and they both contain a C-terminal prenylation box (22,23). Surprisingly, even without the leucine zipper, MYPT3 and PP1c␦ could be shown to form a complex that exists in heterotetrameric form.…”

“…Recently, it was reported that phosphorylation of an adjacent serine residue (Ser-695) in MYPT1 prevented the phosphorylation of Thr-696, adding yet more complexity to the regulation of MYPT1 (20). Apart from these known regulatory subunits, there are several other members of a growing MYPT protein family, including the unconventional myosin Myr8 iso-forms (21) and the closely related MYPT3 and TIMAP (22)(23)(24). However, it is not known if these novel MYPT-related proteins are bona fide targeting subunits of myosin phosphatase with similar phosphorylation regulation mechanisms.…”

“…Interestingly, MYPT3 and TIMAP share similar domain structures that differ from all other MYPT family members (7,23,24). Both have a C-terminal CAAX prenylation motif that targets the protein to cell membranes, similar to that of the Rho GTPases (25).…”

The Foxn1‐dependent transcripts PCOLCE2 and mPPP1R16B are not required for normal thymopoiesis

“…3A, B) and it encodes a protein with predicted protein-protein interaction domains. The foremost reason, however, to study this transcript was the recent demonstration that its expression is regulated by TGF-b signaling [17]. Indeed, there is good evidence that signaling through TGF-b family members is involved in thymus development [18,19].…”

“…Homology of the predicted protein was found to be highest to regulatory subunits of protein phosphatase 1 that target this serine/threonine phosphatase to distinct subcellular locations or specific substrates [20]. Based on sequence homology, the WDT4 gene product has now been annotated as murine protein phosphatase 1 regulatory inhibitory subunit 16B (mPPP1R16B) [17,21]. A functional role has so far not been attributed.…”