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*
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten and
Max-von-Pettenkofer-Institut für Medizinische Mikrobiologie, Universität München, München, Germany; and
Eisai London Research Laboratories, University College, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently identified an additional, Ca2+-independent pathway by which thrombin and Pasteurella multocida toxin can control MLC phosphorylation in HUVEC. This pathway is mediated by the Ras-related GTP-binding protein Rho (3, 4). The ubiquitous Rho-GTPases play a pivotal role in the control of cellular actin rearrangements and cell shape (10). Depending on the cell type, Rho regulates formation of stress fibers and focal adhesion sites or cell contraction (3, 11, 12, 13). In its active GTP-bound form, Rho stimulates the effector molecule p160 Rho kinase which phosphorylates and thereby inactivates MLC phosphatase (14, 15, 16). It is assumed that Rho-mediated inactivation of MLC phosphatase favors MLC phosphorylation in the presence of MLC kinase activity (3, 4). Conversely, when Rho is inhibited, e.g., by C3 transferase, MLC phosphatase activity increases and MLC dephosphorylation is favored. Seemingly through this mechanism, thrombin- and P. multocida toxin-induced MLC phosphorylation and cell contraction can be blocked by Rho inhibitors.
It is well documented that drugs that elevate the intracellular concentration of cAMP prevent vascular leakage induced by inflammatory mediators (17, 18, 19). cAMP seems to inhibit thrombin- and histamine-induced MLC phosphorylation and endothelial cell contraction without affecting the Ca2+ influx induced by these mediators (7, 18). Elevation of cAMP was shown to disrupt the actin cytoskeleton of quiescent endothelial cells and to prevent thrombin-induced reorganization of actin (18, 20). Interestingly, inhibition of Rho by C3 transferase from Clostridium botulinum blocks endothelial contractility in the same manner as cAMP-elevating drugs (3, 18). Moreover, cAMP-elevating agents and C3 transferase can in a similar way inhibit various Rho-mediated cell functions such as tyrosine phosphorylation of paxillin in murine Y1 adrenal cells (21), NK cell function (22), melanoma cell differentiation (23), and integrin receptor clustering (24). Furthermore, it was shown that cAMP-induced morphological changes of neuronal cells are counteracted by active Rho (25). Given these widespread similarities in the cellular effects of cAMP-elevating agents and Rho inhibitors, we reasoned that the inhibitory effect of cAMP on endothelial cell contraction might be due to inhibition of the Rho/Rho kinase signaling pathway. A molecular mechanism by which cAMP could directly inhibit Rho was suggested previously. It was shown that protein kinase A (PKA) can phosphorylate recombinant RhoA on serine 188 in vitro and in intact NK cells (22). However, so far it has not been demonstrated that PKA phosphorylates RhoA in all cell types.
In this study, we provide evidence that in human endothelial cells LPS induces MLC phosphatase deactivation and an increase in MLC phosphorylation via Rho/Rho kinase. In addition, we show that cAMP-elevating substances inhibit Rho/Rho kinase signaling at a level upstream of Rho, leading to a blockage of LPS-induced MLC phosphorylation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
HUVEC were obtained and cultured as described previously (3, 4). Briefly, cells harvested from umbilical cords were plated onto collagen-coated (24 h, 100 µg/ml collagen G; Biochrom, Berlin, Germany) plastic culture flasks and cultured in endothelial growth medium (Promo Cell, Heidelberg, Germany) containing endothelial cell growth supplement/heparin and 10% FCS. For all experiments, cells were plated at a density of 2 x 104 cells/cm2 and grown to confluency for 10 days, with medium changes every 2–3 days.
Immunofluorescence
For fluorescence staining, HUVEC were plated (2 x 104 cells/cm2) on Eppendorf Cellocate glass coverslips (Eppendorf, Hamburg, Germany) coated with 100 µg/ml collagen G (20°C, 24 h) and grown to confluency for 10 days. To label F-actin, cells were fixed for 10 min with 3.7% formaldehyde in PBS containing 1 mM Ca2+ and 1 mM Mg2+, permeabilized for 5 min in cold acetone (-20°C), and air dried. Coverslips were then incubated for 20 min with rhodamine phalloidin (1/20 in PBS; Molecular Probes, Eugene, OR) and mounted in Mowiol (Calbiochem) containing 0.2% p-phenylenediamine (Sigma) as anti-fading agent. For immunostaining of phosphorylated MLC, cells were fixed as above, permeabilized with 0.1% Triton X-100 for 5 min, and then blocked for 30 min with 10% normal goat serum. Coverslips were then incubated with polyclonal rabbit Ab against phosphorylated MLC for 1 h, followed by rhodamine-conjugated goat anti-rabbit Ab. All steps were performed at room temperature with three washes in PBS between Ab incubations. Fluorescence microscopy was performed with a Leica RBM 3 fluorescence microscope (Leica, Bensheim, Germany) and microphotographs were recorded with a digital spot camera (Visitron Systems, Puchheim, Germany).
Recombinant proteins
Recombinant C3 transferase and V14RhoA were expressed as glutathione S-transferase fusion proteins in E. coli and purified on glutathione-Sepharose beads as described elsewhere (3, 4). The fusion proteins were cleaved by thrombin, thrombin was removed by incubation with p-aminobenzamidine beads, and thereafter proteins were concentrated and dialyzed against microinjection buffer (see below). Purity and complete removal of thrombin was checked by SDS-PAGE and Coomassie blue staining. Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) using BSA as standard. As tested by SDS-PAGE and Coomassie blue staining, protein preparations showed essentially only one band.
Microinjection
Microinjection was performed with a Compic Inject micromanipulator (Cell Biology Trading, Hamburg, Germany). Cells were plated and cultured on Cellocate coverslips (Eppendorf) as described above. V14RhoA was diluted with microinjection buffer (150 mM NaCl, 50 mM Tris, 5 mM MgCl2, pH 7.5) and injected at a concentration of 1 µg/µl into the cytoplasm of HUVEC. Control injections conducted with microinjection buffer did not produce any significant effect on cell morphology or actin organization. The microinjected volume was about 1–3 x 10-15 liter/cell. Injected cells were identified by coinjecting rat IgG (5 mg/ml) followed by staining with FITC-conjugated goat anti-rat IgG (Dianova, Hamburg, Germany). For each experiment, at least 100 cells were injected and examined by fluorescence microscopy.
MLC phosphorylation
MLC phosphorylation was analyzed by SDS-PAGE and Western blotting. HUVEC were grown for 10 days in 6-well culture dishes and treated with LPS as indicated. Cells were lysed with boiling Laemmli sample buffer. Proteins were then electroblotted onto polyvinylidene difluoride membranes using Mini Protean II electrophoresis cells (Bio-Rad, Richmond, CA). Membranes were incubated overnight with anti-phospho-MLC Ab (1/500) in Tris-buffered saline containing 0.3% Tween 20, washed three times, incubated for 1 h with HRP-labeled goat anti-rabbit Ab (Amersham, Arlington Heights, IL), 1/7500 in Tris-buffered saline, washed three times, and then developed with luminol solution (Pierce) and exposed to Kodak X-OMAT films (Kodak, Rochester, NY. The amount of phosphorylated MLC was determined by densitometric analysis of the protein bands that reacted with anti-phospho-MLC Ab using a Sharp XL-325 densitometer (Sharp Electronics, Hamburg, Germany) and Pharmacia Image Master software (Pharmacia, Piscataway, NJ). The protein load of MLC was checked by Western blot using anti-MLC Ab (Sigma).
Preparation of myosin-enriched cell fractions
Myosin-enriched fractions of HUVEC were prepared as described previously (3, 4). Briefly, HUVEC were plated on collagen-coated 100-mm diameter plates (Falcon, Becton Dickinson, Plymouth, U.K.) and cultivated for 10 days. Monolayers were washed twice with ice-cold PBS (Sigma) and 200 µl of homogenization buffer [50 mM Tris-aminomethane (pH 7.5), 0.1 mM EDTA, 28 mM 2-ME, 1 µg/ml of leupeptin and pepstatin, Pefabloc, and aprotinin] was added. Plates were incubated at -80°C, scraped with a rubber policeman, and homogenized by passing the suspension several times through a syringe. Homogenates were then treated with high salt buffer (0.6 M NaCl, 0.1% Tween 20, containing 1 µg/ml of leupeptin and pepstatin, aprotinin, and Pefabloc) for 1 h at 4°C and subsequently centrifuged at 4500 x g for 30 min at 4°C. The supernatant was diluted 10-fold with assay buffer (50 mM Tris, 0.1 mM EDTA, and 28 mM 2-ME, pH 7.0) and centrifuged for 30 min at 10,000 x g at 4°C. The resulting pellet was resolved in 10 µl of high salt buffer. This myosin-enriched cell fraction contains mostly protein phosphatase (PP) 1 and essentially no PP2 (4).
Measurement of myosin-associated phosphatase activity
Phosphatase activity was determined in myosin-enriched fractions by using the protein phosphatase assay system (Life Technologies, Rockville, MD) according to the instructions of the manufacturer. This assay system is based on a method described previously (3, 4). Briefly, phosphorylase b was in vitro phosphorylated in the presence of [32P]ATP by phosphorylase kinase to obtain a substrate for PP1 and PP2. Cell homogenates were supplemented with 1 nM (final concentration) okadaic acid to selectively inhibit PP2. PP1 activities of cell homogenates were then quantified by measuring release of radioactivity from [32P]phosphorylase b.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that thrombin and P.
multocida toxin can inhibit MLC phosphatase in HUVEC by a
mechanism involving Rho and its target Rho kinase (3, 4).
We proposed that in concert with
Ca2+/calmodulin-dependent MLC kinase, the lowered
MLC phosphatase activity enhances MLC phosphorylation and
endothelial cell contraction. Endothelial cell contraction is
considered a hallmark of pathologically increased endothelial/vascular
permeability (Fig. 1
). Because it has
been shown that LPS can stimulate endothelial cell contraction in a
Ca2+-independent manner, we asked whether LPS
might signal via the Rho/Rho kinase pathway (8, 9). To
test this possibility, we measured MLC phosphatase activity and MLC
phosphorylation in HUVEC treated with LPS (100 ng/ml) for different
time periods. As indicated in Fig. 2a, LPS stimulation markedly
reduced MLC phosphatase activity in HUVEC, with a minimum of MLC
phosphatase activity reached 1–2 h after LPS addition. MLC phosphatase
down-regulation was followed by an increase in MLC phosphorylation that
reached a maximum 3 h after LPS addition (Fig. 2b). In
endothelial cells pretreated with the Rho inhibitor C3 transferase from
C. botulinum (24 h, 5 µg/ml) or the Rho kinase inhibitor
Y-27632 (30 min, 10 µM), no decrease in MLC phosphatase was
detectable after LPS addition (Fig. 3b). In fact, MLC phosphatase
almost remained at the elevated levels seen in the C3 transferase- or
Y-27632-treated cells (Fig. 3a). Likely as a consequence
of this in the C3 transferase- or Y-27632-pretreated cells, MLC
phosphorylation stimulated by LPS was abolished (Fig. 4). Together these findings suggest that
LPS inactivates MLC phosphatase via Rho/Rho kinase which produces
enhanced MLC phosphorylation. Furthermore, elevation of MLC phosphatase
by C3 transferase or Y-27632 treatment indicates that a basal Rho/Rho
kinase activity keeps MLC phosphatase at an intermediate level in
unstimulated endothelial cells. This might be advantageous because an
intermediate level of MLC phosphatase can rapidly be altered in both
directions: increased by Rho inhibition or decreased by Rho
activation.
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Given that elevation of intracellular cAMP can prevent endothelial
cell contraction stimulated by LPS (8, 9), we asked
whether the underlying mechanism might be inhibition of the Rho/Rho
kinase pathway. To test this notion, endothelial cells were treated
with combinations of cholera toxin (CTx) and isobutyl-methyl-xanthin
(IBMX) or forskolin and dibutyryl cAMP to maximally elevate
intracellular cAMP levels (17, 18). In such treated
endothelial cells, MLC phosphatase increased to a similar degree as
after C3 transferase or Y-27632 treatment (Fig. 3
a). In
addition, CTx/IBMX or forskolin/dibutyryl cAMP treatment inhibited the
LPS-induced decrease in MLC phosphatase (Fig. 3b), and
furthermore these substances blocked MLC phosphorylation stimulated by
LPS in a manner similar to C3 transferase or Y-27632 (Fig. 4). These
data strongly suggest that cAMP elevation inhibits the Rho/Rho kinase
pathway, leading to increased MLC phosphatase activity and a blockage
of MLC phosphorylation. To test whether the inhibitory cAMP effect on
MLC phosphorylation is mediated by PKA, we treated cells with the
highly selective and cell-permeable PKA-inhibiting peptide PKI-1422 (1
h, 40 nM) or with the PKA inhibitor H89 (1 h, 50 nM). If the inhibitory
cAMP effect on LPS-induced MLC phosphorylation was mediated by PKA, it
should be blockable by the PKA inhibitors. As indicated in Fig. 4, however, inhibition of LPS-induced MLC phosphorylation by cAMP could
not be prevented by the PKA inhibitors, suggesting a PKA-independent
mechanism.
cAMP elevation does not induce phosphorylation of Rho in HUVEC
We could confirm that recombinant RhoA is a good substrate for PKA
in vitro whereas a serine 188 to alanine Rho mutant was not
phosphorylated (data not shown). To clarify whether phosphorylation of
Rho takes place in intact HUVEC, we used two-dimensional gel
electrophoresis because we failed to precipitate significant amounts of
Rho with a variety of commercially available anti-RhoA Abs.
Recombinant Rho, like many proteins, shows an acidic shift in
two-dimensional gels upon phosphorylation due to the additional
negative charge of the phosphate group (M. Aepfelbacher and M. Essler,
unpublished observations). As demonstrated in Fig. 5a, RhoA in HUVEC appeared as
two spots with pI values of 5.9 and 6.3. respectively, in
two-dimensional gels. The nature of these two RhoA spots is not known,
but in CTx/IBMX-treated cells none of the two RhoA spots shifted to a
more acidic pH (Fig. 5b). To further test for
phosphorylation of Rho, we labeled CTx/IBMX-treated HUVEC with
inorganic [32P]phosphate, separated
phosphorylated proteins with two-dimensional gel electrophoresis, and
blotted them onto a polyvinylidene difluoride membrane. The blot
membrane was first subjected to autoradiography and then to
anti-RhoA immunoblot. Although dozens of phosphorylated proteins
were detected in CTx/IBMX-treated HUVEC, no phosphorylated protein
comigrated with the two RhoA spots found in Western blot (Fig. 5, c and d). Altogether these data suggest that RhoA
is not phosphorylated in HUVEC after elevation of cAMP.
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To determine more exactly at which level of the Rho/Rho kinase
signaling pathway cAMP might act, we microinjected constitutively
active V14Rho or inhibited MLC phosphatase with the drug calyculin A
(15 min, 10 nM) in cells pretreated with CTx/IBMX. Thereafter, cells
were stained with an Ab recognizing the phosphorylated forms of MLC
only. Control cells showed almost no phosphorylated MLC (Fig. 6a). LPS treatment stimulated
the appearance of phosphorylated myosin along stress fibers (Fig. 6b). Pretreatment of cells with CTx/IBMX prevented
LPS-induced MLC phosphorylation and stress fiber formation (Fig. 6c). Microinjection of V14Rho (Fig. 6d) caused
cell contraction and MLC phosphorylation, mimicking the effect of LPS.
However, this effect was not inhibited by pretreatment of cells with
CTx/IBMX (Fig. 6e). Furthermore, treatment with calyculin A
caused the appearance of phosphorylated MLC along stress fibers (Fig. 6f) but this could also not be inhibited by CTx/IBMX (Fig. 6g). Taken together, these data indicate that cAMP inhibits
Rho/Rho kinase signaling by acting on an upstream regulator of
Rho.
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It is well documented that LPS enhances protein synthesis in
endothelial cells by activation of transcription and translation. The
newly formed proteins are partly secreted and can stimulate endothelial
cells in a paracrine manner. Therefore, LPS-induced MLC phosphorylation
could be due to activation of Rho/Rho kinase by secreted mediators like
IL-6 (27). To test whether de novo synthesis of protein is
required for LPS-induced MLC phosphorylation, we stimulated HUVEC with
LPS (3 h, 100 ng/ml) in the presence of cycloheximide (10 µM) or
actinomycin D (10 µM) to inhibit translation or transcription,
respectively. As indicated by Fig. 7,
both inhibitors blocked LPS-induced MLC phosphorylation, suggesting
that MLC phosphorylation after LPS stimulation occurs secondary to
protein synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS signaling in CD14-bearing cells such as monocytes and macrophages
has been well characterized, while less is known about signal pathways
stimulated by LPS in non-CD14-bearing cells such as human endothelial
cells. It has been shown that activation of endothelial cells by LPS is
dependent on soluble CD14 (sCD14) present in serum, which binds LPS to
form a LPS-sCD14 complex (27). It is unclear which
receptors are activated by the LPS-sCD14 complex and which second
messengers these hypothetical receptors stimulate. Hence, the signal
pathway by which LPS activates Rho in endothelial cells still remains
to be investigated. It was suggested that heterotrimeric G
protein-coupled receptors can activate Rho through p115 Rho GEF, a
molecule that acts both as a GTPase-activating protein for G
subunits and a nucleotide exchange factor for Rho (28). In
fact, involvement of heterotrimeric G proteins in CD14-dependent LPS
signaling was suggested recently (29). Another report
suggested that the epidermal growth factor-receptor tyrosine kinase
is an intermediary step between cell surface receptors and Rho
activation (30). Interestingly, tyrosine kinase
inhibitors such as genistein and herbimycin A were recently found to
attenuate LPS effects (16).
Because stimulation of Rho/Rho kinase signaling starts not before 1 h after LPS addition and can be inhibited by a transcription and a translation inhibitor, it seems that Rho activation is due to secondary mediators secreted by endothelial cells. For instance, IL-6 was shown to be secreted by endothelial cells subsequent to LPS stimulation (27). Therefore, LPS-induced mediators such as IL-6 could activate Rho in a paracrine manner. Whatever the upstream mechanisms are, we clearly demonstrate here that activation of Rho/Rho kinase and inactivation of MLC phosphatase is a consequence of presentation of LPS to endothelial cells. LPS activates mitogen-activated protein kinase by a sCD14-dependent mechanism in HUVEC within minutes (31). One additional explanation for the time lag between LPS stimulation and MLC phosphorylation in endothelial cells could be that an increase in MLC phosphorylation requires a long-term inactivation (minutes to hours) of MLC phosphatase to become significant.
Recent studies have highlighted the importance of intracellular cAMP concentration as a gating element in regulation of endothelial cell contraction and vascular permeability (7, 17, 18). Here, we report that cAMP-elevating drugs inhibit LPS-induced inactivation of MLC phosphatase and subsequent MLC phosphorylation. Furthermore, these drugs enhance MLC phosphatase activity in a manner similar to inhibitors of Rho or Rho kinase, suggesting that cAMP elevation inhibits basal Rho/Rho kinase activity in human endothelial cells. Because cAMP elevation does not interfere with MLC phosphorylation induced by constitutive active Rho or calyculin A, we conclude that cAMP blocks an upstream RhoA regulator. cAMP elevation might, for example, interfere with GDP/GTP exchange on Rho as proposed to occur in mouse lymphocyte cell lines (32). Interestingly it was reported recently that cAMP regulates the activity of the rap guanine-nucleotide exchange factor Epac independently of PKA (33). Therefore, it could be speculated that cAMP utilizes another PKA-independent mechanism to inhibit the GDP/GTP exchange on Rho. It was shown that cAMP-dependent protein kinase A phosphorylates recombinant RhoA on serine 188 and that as a consequence Rho in intact cells might be inactivated (22). However, by two-dimensional gel analysis, we could not find any indication that RhoA is phosphorylated in endothelial cells. Furthermore, the effect of cAMP-elevating substances was not overcome by inhibition of PKA, suggesting that cAMP interferes with Rho signaling by a PKA-independent mechanism. It is well documented that the small GTP-binding protein Rac activates or inactivates Rho depending on the cell type (10, 34). Therefore, inactivation of Rho could also be caused by cAMP-mediated modulation of Rac activity. In this context, it was found that the cAMP-induced differentiation of melanoma cells is not only overcome by transfection of cells with constitutively active Rho, but also by transfection with constitutively active Rac (23). In any case, the direct target of cAMP in endothelial cells controlling Rho activity awaits further identification.
The Rho/Rho kinase pathway seems to be a crucial checkpoint for regulation of endothelial permeability in vitro and in vivo. Therefore, it could be envisioned that one therapeutical strategy during septic shock consists in the blockage of Rho/Rho kinase to prevent vascular leakage. cAMP-elevating drugs could be potentially useful for this purpose at present and the newly available Rho kinase inhibitors might be an option for the future.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Markus Essler, Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstrasse 9, 80336 München, Germany.
3 Address corresondence and reprint requests to Dr. Martin Aepfelbacher, Max-von-Pettenkofer-Institut, Pettenkoferstrasse 9a, 80336 München, Germany.
4 Abbreviations used in this paper: MLC, myosin light chain; CTx, cholera toxin; IBMX, isobutyl-methyl-xanthin; PKA, protein kinase A; sCD14, soluble CD14; PP, protein phosphatase.
Received for publication April 28, 1999. Accepted for publication April 5, 2000.
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is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol. Cell. Biol. 16:5313.[Abstract]
. J. Biol. Chem. 273:22554.13. Science 280:2112.This article has been cited by other articles:
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A. P. SOMLYO and A. V. SOMLYO Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase Physiol Rev, October 1, 2003; 83(4): 1325 - 1358. [Abstract] [Full Text] [PDF]
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M. Aepfelbacher, C. Trasak, G. Wilharm, A. Wiedemann, K. Trulzsch, K. Krauss, P. Gierschik, and J. Heesemann Characterization of YopT Effects on Rho GTPases in Yersinia enterocolitica-infected Cells J. Biol. Chem., August 29, 2003; 278(35): 33217 - 33223. [Abstract] [Full Text] [PDF]
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J. Qiao, F. Huang, and H. Lum PKA inhibits RhoA activation: a protection mechanism against endothelial barrier dysfunction Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L972 - L980. [Abstract] [Full Text] [PDF]
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 V. D. Dontchev and P. C. Letourneau Growth Cones Integrate Signaling from Multiple Guidance Cues J. Histochem. Cytochem., April 1, 2003; 51(4): 435 - 444. [Abstract] [Full Text] [PDF]
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 M. C.P. Glyn, J. G. Lawrenson, and B. J. Ward A Rho-associated kinase mitigates reperfusion-induced change in the shape of cardiac capillary endothelial cells in situ Cardiovasc Res, January 1, 2003; 57(1): 195 - 206. [Abstract] [Full Text] [PDF]
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S. Hippenstiel, M. Witzenrath, B. Schmeck, A. Hocke, M. Krisp, M. Krull, J. Seybold, W. Seeger, W. Rascher, H. Schutte, et al. Adrenomedullin Reduces Endothelial Hyperpermeability Circ. Res., October 4, 2002; 91(7): 618 - 625. [Abstract] [Full Text] [PDF]
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 L.-Y. Chen, B. L. Zuraw, F.-T. Liu, S. Huang, and Z. K. Pan IL-1 Receptor-Associated Kinase and Low Molecular Weight GTPase RhoA Signal Molecules Are Required for Bacterial Lipopolysaccharide-Induced Cytokine Gene Transcription J. Immunol., October 1, 2002; 169(7): 3934 - 3939. [Abstract] [Full Text] [PDF]
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T. Gudi, J. C. Chen, D. E. Casteel, T. M. Seasholtz, G. R. Boss, and R. B. Pilz cGMP-dependent Protein Kinase Inhibits Serum-response Element-dependent Transcription by Inhibiting Rho Activation and Functions J. Biol. Chem., September 27, 2002; 277(40): 37382 - 37393. [Abstract] [Full Text] [PDF]
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 V. D. Dontchev and P. C. Letourneau Nerve Growth Factor and Semaphorin 3A Signaling Pathways Interact in Regulating Sensory Neuronal Growth Cone Motility J. Neurosci., August 1, 2002; 22(15): 6659 - 6669. [Abstract] [Full Text] [PDF]
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Y. Jin, S. J. Atkinson, J. A. Marrs, and P. J. Gallagher Myosin II Light Chain Phosphorylation Regulates Membrane Localization and Apoptotic Signaling of Tumor Necrosis Factor Receptor-1 J. Biol. Chem., August 3, 2001; 276(32): 30342 - 30349. [Abstract] [Full Text] [PDF]
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