Raphaela Schwappacher

Modulation of dynamin-related protein 1 (DRP1) function by increased O-linked-β-N-acetylglucosamine modification (O-GlcNAc) in cardiac myocytes

Background: DRP1 plays a significant role to control mitochondrial fission. Results: DRP1 is O-GlcNAcylated. Increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from cytoplasm to mitochondria. Conclusion: O-GlcNAcylation modulates DRP1 function, which has consequences for mitochondrial function. Significance: The modulation of DRP1 function by increased overall O-GlcNAcylation could play a significant role in the development of diabetic mitochondrial dysfunction. O-linked-N-acetyl-glucosamine glycosylation (O-GlcNAcylation) of the serine and threonine residues of cellular proteins is a dynamic process and affects phosphorylation. Prolonged O-GlcNAcylation has been linked to diabetes-related complications, including mitochondrial dysfunction. Mitochondria are dynamically remodeling organelles, that constantly fuse (fusion) and divide (fission). An imbalance of this process affects mitochondrial function. In this study, we found that dynamin-related protein 1 (DRP1) is O-GlcNAcylated in cardiomyocytes at threonine 585 and 586. O-GlcNAcylation was significantly enhanced by the chemical inhibition of N-acetyl-glucosaminidase. Increased O-GlcNAcylation decreases the phosphorylation of DRP1 at serine 637, which is known to regulate DRP1 function. In fact, increased O-GlcNAcylation augments the level of the GTP-bound active form of DRP1 and induces translocation of DRP1 from the cytoplasm to mitochondria. Mitochondrial fragmentation and decreased mitochondrial membrane potential also accompany the increased O-GlcNAcylation. In conclusion, this report shows, for the first time, that O-GlcNAcylation modulates DRP1 functionality in cardiac muscle cells.

Nongenomic Thyroid Hormone Signaling Occurs Through a Plasma Membrane–Localized Receptor

Signaling from a plasma membrane–associated receptor contributes to the effects of thyroid hormones on bones. Rapidly Promoting Bone Growth from the Membrane Thyroid hormones regulate many processes, including bone turnover. By entering cells and binding to nuclear receptors, thyroid hormones induce target gene expression; however, they also stimulate rapid cellular changes that are independent of gene regulation. Kalyanaraman et al. found an alternative form of the thyroid receptor that associated with the cellular plasma membrane of bone cells. Stimulation of this receptor by thyroid hormones increased the numbers of bone cells and protected them from death. Treatment of mice deficient in thyroid hormones with a compound that mimicked signaling from this membrane-associated receptor reversed defects in bone formation, suggesting that this form of thyroid hormone action may be clinically relevant. Thyroid hormone (TH) is essential for vertebrate development and the homeostasis of most adult tissues, including bone. TH stimulates target gene expression through the nuclear thyroid receptors TRα and TRβ; however, TH also has rapid, transcription-independent (nongenomic) effects. We found a previously uncharacterized plasma membrane–bound receptor that was necessary and sufficient for nongenomic TH signaling in several cell types. We determined that this receptor is generated by translation initiation from an internal methionine of TRα, which produces a transcriptionally incompetent protein that is palmitoylated and associates with caveolin-containing plasma membrane domains. TH signaling through this receptor stimulated a pro-proliferative and pro-survival program by increasing the intracellular concentrations of calcium, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), which led to the sequential activation of protein kinase G II (PKGII), the tyrosine kinase Src, and extracellular signal–regulated kinase (ERK) and Akt signaling. Hypothyroid mice exhibited a cGMP-deficient state with impaired bone formation and increased apoptosis of osteocytes, which was rescued by a direct stimulator of guanylate cyclase. Our results link nongenomic TH signaling to a previously uncharacterized membrane-bound receptor, and identify NO synthase, guanylate cyclase, and PKGII as TH effectors that activate kinase cascades to regulate cell survival and proliferation.

Cyclic GMP and Protein Kinase G Control a Src-Containing Mechanosome in Osteoblasts

Drugs that activate protein kinase G could mimic the bone-building effects of mechanical stimulation. Building Bone The loss of bone density that afflicts individuals with osteoporosis makes them more vulnerable to bone fractures. One way to counteract decreases in bone density is through exercise, which mechanically stimulates bone tissue and initiates proliferation in bone-forming cells. Alternatively, the signaling pathways that mediate this proliferative response could be therapeutically activated to mimic the effects of mechanical stimulation. Nitric oxide (NO) is a second messenger that is produced in response to mechanical stimulation; it triggers production of cyclic GMP (cGMP) and, consequently, activation of protein kinase G (PKG). Rangaswami et al. delineated a pathway in mechanically stimulated osteoblasts whereby activation of PKGII signaling ultimately leads to a proliferative response. Mechanical stimuli triggered the formation of a complex containing PKGII, the tyrosine kinase Src, the phosphatases SHP-1 and SHP-2, and β3 integrin mechanoreceptors. Activation of Src in this complex led to activation of extracellular signal–regulated kinase (ERK), which in turn elicited changes in gene expression that promote proliferation. Thus, PKG-activating drugs could be used to mimic the anabolic effects of mechanical stimulation on bone in the treatment of osteoporosis. The accompanying Perspective by Bidwell and Pavalko describes other examples of signaling pathways that mediate mechanotransduction in bone cells. Mechanical stimulation is crucial for bone growth and remodeling, and fluid shear stress promotes anabolic responses in osteoblasts through multiple second messengers, including nitric oxide (NO). NO triggers production of cyclic guanosine 3′,5′-monophosphate (cGMP), which in turn activates protein kinase G (PKG). We found that the NO-cGMP-PKG signaling pathway activates Src in mechanically stimulated osteoblasts to initiate a proliferative response. PKGII was necessary for Src activation, a process that also required the interaction of Src with β3 integrins and dephosphorylation of Src by a complex containing the phosphatases SHP-1 (Src homology 2 domain–containing tyrosine phosphatase 1) and SHP-2. PKGII directly phosphorylated and stimulated SHP-1 activity, and fluid shear stress triggered the recruitment of PKGII, Src, SHP-1, and SHP-2 to a mechanosome containing β3 integrins. PKGII-null mice showed defective Src and ERK (extracellular signal–regulated kinase) signaling in osteoblasts and decreased ERK-dependent gene expression in bone. Our findings reveal a convergence of NO-cGMP-PKG and integrin signaling and establish a previously unknown mechanism of Src activation. These results support the use of PKG-activating drugs to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Novel crosstalk to BMP signalling: cGMP-dependent kinase I modulates BMP receptor and Smad activity

Integration of multiple signals into the canonical BMP/Smad pathway poses a big challenge during the course of embryogenesis and tissue homeostasis. Here, we show that cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent kinase I (cGKI) modulates BMP receptors and Smads, providing a novel mechanism enhancing BMP signalling. cGKI, a key mediator of vasodilation and hypertension diseases, interacts with and phosphorylates the BMP type II receptor (BMPRII). In response to BMP‐2, cGKI then dissociates from the receptors, associates with activated Smads, and undergoes nuclear translocation. In the nucleus, cGKI binds with Smad1 and the general transcription factor TFII‐I to promoters of BMP target genes such as Id1 to enhance transcriptional activation. Accordingly, cGKI has a dual function in BMP signalling: (1) it modulates BMP receptor/Smad activity at the plasma membrane and (2) after redistribution to the nucleus, it further regulates transcription as a nuclear co‐factor for Smads. Consequently, cellular defects caused by mutations in BMPRII, found in pulmonary arterial hypertension patients, were compensated through cGKI, supporting the positive action of cGKI on BMP‐induced Smad signalling downstream of the receptors.

PP2A regulates BMP signalling by interacting with BMP receptor complexes and by dephosphorylating both the C-terminus and the linker region of Smad1.

Phosphorylation of Smads is a crucial regulatory step in the signal transduction pathway initiated by bone morphogenetic proteins (BMPs). Although the dephosphorylation events terminating the pathway in the nucleus have been characterized, little is known about the dephosphorylation of Smads in the cytoplasm. In a proteomic screen for proteins interacting with the BMP type-II receptor, we found the regulatory Bβ subunit of PP2A. PP2A is one of the major serine/threonine phosphatases involved in cell-cycle regulation and signal transduction. Here, we present data showing that the Bβ subunit of PP2A interacts with both BMP type-I and type-II receptors. Furthermore, we demonstrate that several B subunits can associate with the BMP type-II receptor, independently of the kinase activity of the receptor and the catalytic subunit of PP2A. By contrast, the PP2A catalytic subunit is required for PP2A function at the receptor complex. This function of PP2A is the dephosphorylation of Smad1, mainly in the linker region. PP2A-mediated dephosphorylation of the BMP-Smad linker region leads to increased nuclear translocation of Smads and overall amplification of the BMP signal. Although other phosphatases identified within the BMP pathway are all shown to inhibit signalling, PP2A is the first example for a signalling stimulatory phosphatase within this pathway.

Protein Kinase G and Focal Adhesion Kinase Converge on Src/Akt/β-Catenin Signaling Module in Osteoblast Mechanotransduction

Background: Fluid shear increases intracellular calcium and NO/cGMP signaling in osteoblasts. Results: Focal adhesion kinase and protein kinase G are independently activated downstream of calcium; both kinases cooperatively activate Src, leading to Akt/GSK3/β-catenin signaling in shear-stressed osteoblasts. Conclusion: Osteoblast mechanotransduction requires cross-talk between FAK and PKG to regulate Akt. Significance: cGMP-elevating agents may prove useful for the treatment of osteoporosis. Mechanical loading of bone induces interstitial fluid flow, leading to fluid shear stress (FSS) of osteoblasts. FSS rapidly increases the intracellular calcium concentration ([Ca2+]) and nitric oxide (NO) synthesis in osteoblasts and activates the protein kinase Akt. Activated Akt stimulates osteoblast proliferation and survival, but the mechanism(s) leading to Akt activation is not well defined. Using pharmacological and genetic approaches in primary human and mouse osteoblasts and mouse MC3T3 osteoblast-like cells, we found that Akt activation by FSS occurred through two parallel pathways; one required calcium stimulation of NO synthase and NO/cGMP/protein kinase G II-dependent activation of Src, and the other required calcium activation of FAK and Src, independent of NO. Both pathways cooperated to increase PI3K-dependent Akt phosphorylation and were necessary for FSS to induce nuclear translocation of β-catenin, c-fos, and cox-2 gene expression and osteoblast proliferation. These data explain how mechanical stimulation of osteoblasts leads to increased signaling through a growth regulatory pathway essential for maintaining skeletal integrity.

Rho isoform-specific interaction with IQGAP1 promotes breast cancer cell proliferation and migration.

Background: RhoA/C and RhoB have homologous sequences, but opposing functions. Results: IQGAP1 binds prenylated, active RhoA/C, but not RhoB; IQGAP1 increases RhoA/C GTP loading and is required for RhoA/C-induced proliferation and motility of breast cancer cells. Conclusion: IQGAP1 is a regulator and pro-oncogenic effector of RhoA/C. Significance: Disrupting Rho/IQGAP interactions with prenylation inhibitors may be a useful adjunct for breast cancer treatment. We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoAL63 and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoAL63-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoCV14 mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.

Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity

Abstract Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

A molecular mechanism for therapeutic effects of cGMP-elevating agents in pulmonary arterial hypertension

Background: Pulmonary arterial hypertension (PAH) is characterized by abnormal vascular remodeling and impaired BMP/Smad signaling. Results: cGMP/PKGI are required for the anti-proliferative and pro-differentiation effects of BMP in pulmonary artery smooth muscle cells (PASMCs) in vivo. Conclusion: Cooperative cGMP and BMP signaling is essential for maintaining a low proliferative, differentiated PASMC phenotype. Significance: These data explain therapeutic cGMP effects in PAH. Pulmonary arterial hypertension (PAH) is a progressive, usually fatal disease with abnormal vascular remodeling. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients are hyperproliferative and apoptosis-resistant and demonstrate decreased signaling in response to bone morphogenetic proteins (BMPs). Cyclic GMP-elevating agents are beneficial in PAH, but their mechanism(s) of action are incompletely understood. Here we show that BMP signaling via Smad1/5/8 requires cGMP-dependent protein kinase isotype I (PKGI) to maintain PASMCs in a differentiated, low proliferative state. BMP cooperation with cGMP/PKGI was crucial for transcription of contractile genes and suppression of pro-proliferative and anti-apoptotic genes. Lungs from mice with low or absent PKGI (Prkg1+/− and Prkg1−/− mice) exhibited impaired BMP signaling, decreased contractile gene expression, and abnormal vascular remodeling. Conversely, cGMP stimulation of PKGI restored defective BMP signaling in rats with hypoxia-induced PAH, consistent with cGMP-elevating agents reversing vascular remodeling in this PAH model. Our results provide a mechanism for the therapeutic effects of cGMP-elevating agents in PAH and suggest that combining them with BMP mimetics may provide a novel, disease-modifying approach to PAH therapy.

cGMP-dependent protein kinase Iβ regulates breast cancer cell migration and invasion via interaction with the actin/myosin-associated protein caldesmon.

Summary The two isoforms of type I cGMP-dependent protein kinase (PKGI&agr; and PKGI&bgr;) differ in their first ∼100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The dimerization domains form coiled-coil structures and serve as platforms for isoform-specific protein–protein interactions. Using the PKGI&bgr; dimerization domain as an affinity probe in a proteomic screen, we identified the actin/myosin-associated protein caldesmon (CaD) as a PKGI&bgr;-specific binding protein. PKGI&bgr; phosphorylated human CaD on serine 12 in vitro and in intact cells. Phosphorylation on serine 12 or mutation of serine 12 to glutamic acid (S12E) reduced the interaction between CaD and myosin IIA. Because CaD inhibits myosin ATPase activity and regulates cell motility, we examined the effects of PKGI&bgr; and CaD on cell migration and invasion. Inhibition of the NO/cGMP/PKG pathway reduced migration and invasion of human breast cancer cells, whereas PKG activation enhanced their motility and invasion. siRNA-mediated knockdown of endogenous CaD had pro-migratory and pro-invasive effects in human breast cancer cells. Reconstituting cells with wild-type CaD slowed migration and invasion; however, CaD containing a phospho-mimetic S12E mutation failed to reverse the pro-migratory and pro-invasive activity of CaD depletion. Our data suggest that PKGI&bgr; enhances breast cancer cell motility and invasive capacity, at least in part, by phosphorylating CaD. These findings identify a pro-migratory and pro-invasive function for PKGI&bgr; in human breast cancer cells, suggesting that PKGI&bgr; is a potential target for breast cancer treatment.