Bone morphogenetic proteins (BMPs) regulate multiple cellular processes, including cell differentiation and migration. Their signals are transduced by the kinase receptors BMPR-I and BMPR-II, leading to Smad transcription factor activation via BMPR-I. LIM kinase (LIMK) 1 is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin depolymerizing factor. During a search for LIMK1-interacting proteins, we isolated clones encompassing the tail region of BMPR-II. Although the BMPR-II tail is not involved in BMP signaling via Smad proteins, mutations truncating this domain are present in patients with primary pulmonary hypertension (PPH). Further analysis revealed that the interaction between LIMK1 and BMPR-II inhibited LIMK1's ability to phosphorylate cofilin, which could then be alleviated by addition of BMP4. A BMPR-II mutant containing the smallest COOH-terminal truncation described in PPH failed to bind or inhibit LIMK1. This study identifies the first function of the BMPR-II tail domain and suggests that the deregulation of actin dynamics may contribute to the etiology of PPH.

3-Phosphoinositide-dependent protein kinase 1 (PDK-1) phosphorylates and activates members of the AGC protein kinase family and plays an important role in the regulation of cell survival, differentiation, and proliferation. However, how PDK-1 is regulated in cells remains elusive. In this study, we demonstrated that PDK-1 can shuttle between the cytoplasm and nucleus. Treatment of cells with leptomycin B, a nuclear export inhibitor, results in a nuclear accumulation of PDK-1. PDK-1 nuclear localization is increased by insulin, and this process is inhibited by pretreatment of cells with phosphatidylinositol 3-kinase (PI3-kinase) inhibitors. Consistent with the idea that PDK-1 nuclear translocation is regulated by the PI3-kinase signaling pathway, PDK-1 nuclear localization is increased in cells deficient of PTEN (phosphatase and tensin homologue deleted on chromosome 10). Deletion mapping and mutagenesis studies unveiled that presence of a functional nuclear export signal (NES) in mouse PDK-1 located at amino acid residues 382 to 391. Overexpression of constitutively nuclear PDK-1, which retained autophosphorylation at Ser-244 in the activation loop in cells and its kinase activity in vitro, led to increased phosphorylation of the predominantly nuclear PDK-1 substrate p70 S6K␤I. However, the ability of constitutively nuclear PDK-1 to induce anchorage-independent growth and to protect against UV-induced apoptosis is greatly diminished compared with the wildtype enzyme. Taken together, these findings suggest that nuclear translocation may be a mechanism to sequestrate PDK-1 from activation of the cytosolic signaling pathways and that this process may play an important role in regulating PDK-1-mediated cell signaling and function.T he 3Ј-phosphoinositide-dependent protein kinase-1 (PDK-1) is a 64-kDa protein comprised of a Ser͞Thr kinase domain near the N terminus and a C-terminal pleckstrin homology (PH) domain (1). This pivotal kinase plays a crucial role in mediating signal transduction downstream of phosphatidylinositol 3-kinase (PI3-kinase) in response to mitogen stimulation. Growth factor stimulation activates PI3-kinase, which converts phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] to phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ]. This lipid product is bound by the PH domain of PDK-1, resulting in the recruitment of PDK-1 to the plasma membrane. Protein kinase B (PKB), the best characterized substrate of PDK-1, is recruited to the plasma membrane when its own PH domain binds to PtdIns(3,4,5)P 3 , leading to colocalization of these two proteins and phosphorylation of PKB by PDK-1 on Thr-308 (1), resulting in PKB activation. Downstream substrates of PKB include the antiapoptotic protein BAD and Forkhead (FH) transcription factors. In addition to PKB, PDK-1 has also been reported to phosphorylate many other kinases, including ribosomal p70 protein kinases, that are involved in the regulation of protein translation (2-5).At the cellular level, PDK-1 regulates key insulin effects such a...

Grb10 is a member of a superfamily of adaptor proteins that includes Grb7 and Grb14. This family of proteins shares a common overall structure, including an N-terminal region harboring a conserved proline-rich motif, a central Pleckstrin homology (PH) domain, a C-terminal Src homology 2 (SH2) domain, and a conserved region located between the PH and the SH2 domains (BPS). Grb10 directly interacts with a number of mitogenic receptor tyrosine kinases including the insulin (IR) and insulin-like growth factor-I (IGF-IR) receptor. Grb10 binds to the regulatory kinase loop of the insulin receptor (IR) via its SH2 and BPS domains. In addition to receptor tyrosine kinases, Grb10 has also been found to interact with non-receptor tyrosine kinases such as Tec and Bcr-Abl, and other cellular signaling molecules such as Raf-1 and the mitogen activated protein (MAP) kinase kinase, MEK. Overexpression of Grb10 has been shown to inhibit or stimulate insulin/IGF-I signaling depending on the expression levels of the specific isoforms, specific cell context, and/or physiologic endpoint. Genetic imprinting of Grb10 has been linked to the congenital disease, Silver-Russell syndrome, which is characterized by pre- and post-natal growth deficiency. This data suggests that Grb10 may function during embryogenesis in regulating insulin/IGF-I signaling as these growth factors play important roles during development. A role of Grb10 as a potent growth inhibitor during was implicated when disruption of the mGrb10 gene in mice resulted in overgrowth of mutant embryos and neonates. Grb10 is expressed in the central nervous system of mice and rats, which suggests that this protein may regulate neuronal insulin signaling and energy metabolism, consistent with its reported role in metabolic insulin action in fat and muscle cells. An important area of future investigation will be to elucidate the mechanism underlying Grb10's ability to regulate peptide hormone action including insulin/IGF-I signaling and to study the physiological role of this adaptor protein in cellular and animal models.

Mutations in the tumor suppressor protein PTEN (phosphatase and tensin homologue deleted on chromosome 10) enhance cell migration, yet the underlying molecular mechanisms remain largely uncharacterized. Loss of PTEN in mouse embryonic fibroblasts (MEFs) correlates with striking cortical actin accumulation. However, how loss of PTEN leads to cortical actin formation and whether the presence of cortical actin contributes to the increased cell migration are unclear. Here we show that overexpression of dominant-negative forms of (DN) PTEN, RhoA or its kinase-dead (KD) effector, PKN, inhibited cortical actin formation, indicating that cortical actin of Pten À/À MEFs is mediated by the PTEN/Rho/PKN pathway. However, neither DN RhoA nor KD PKN inhibited the enhanced migration of Pten À/À cells, in contrast to the inhibitory effect of DN Rac. In agreement with the previous observation that DN Akt inhibits migration of Pten À/À cells, we demonstrate here that overexpression of KD PDK-1, the Akt kinase, reduces Pten À/À cell migration. Furthermore, overexpression of DN forms of Akt, Rac, or PDK-1, all of which inhibit migration of Pten À/À cells, had no effect on cortical actin accumulation. Our findings suggest that PDK-1/Akt signaling pathway plays a major role in regulating cell migration induced by PTEN deficiency.

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