Myosin phosphatase and cofilin mediate cAMP/cAMP-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin II regulatory light chain phosphorylation

Department of Pathology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 10/2005; 280(38):33083-95. DOI: 10.1074/jbc.M503173200
Source: PubMed

ABSTRACT This study determined the effects of increased intracellular cAMP and cAMP-dependent protein kinase activation on endothelial cell basal and thrombin-induced isometric tension development. Elevation of cAMP and maximal cAMP-dependent protein kinase activation induced by 10 microm forskolin, 40 microm 3-isobutyl-1-methylxanthine caused a 50% reduction in myosin II regulatory light chain (RLC) phosphorylation and a 35% drop in isometric tension, but it did not inhibit thrombin-stimulated increases in RLC phosphorylation and isometric tension. Elevation of cAMP did not alter myosin light chain kinase catalytic activity. However, direct inhibition of myosin light chain kinase with KT5926 resulted in a 90% decrease in RLC phosphorylation and only a minimal decrease in isometric tension, but it prevented thrombin-induced increases in RLC phosphorylation and isometric tension development. We showed that elevated cAMP increases phosphorylation of RhoA 10-fold, and this is accompanied by a 60% decrease in RhoA activity and a 78% increase in RLC phosphatase activity. Evidence is presented that it is this inactivation of RhoA that regulates the decrease in isometric tension through a pathway involving cofilin. Activated cofilin correlates with increased F-actin severing activity in cell extracts from monolayers treated with forskolin/3-isobutyl-1-methylxanthine. Pretreatment of cultures with tautomycin, a protein phosphatase type 1 inhibitor, blocked the effect of cAMP on 1) the dephosphorylation of cofilin, 2) the decrease in RLC phosphorylation, and 3) the decrease in isometric tension. Together, these data provide in vivo evidence that elevated intracellular cAMP regulates endothelial cell isometric tension and RLC phosphorylation through inhibition of RhoA signaling and its downstream pathways that regulate myosin II activity and actin reorganization.

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    • "Naseem's group showed that PKA inhibits ROCK phosphorylation and activation by phosphorylation/ inhibition of RhoA on serine-188 (Aburima et al. 2013). Other studies support this idea by showing that elevated cAMP levels may indirectly lead to cofilin dephosphorylation (Goeckeler and Wysolmerski 2005; Meberg et al. 1998). On the other hand, another study showed that LIMK is directly activated by PKA through phosphorylation on serine-323 and -596 (Nadella et al. 2009). "
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    ABSTRACT: The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.
    Cell and Tissue Research 06/2015; DOI:10.1007/s00441-015-2229-1 · 3.33 Impact Factor
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    • "It is well known from studies of endothelial cells that endothelial contractility results from activation of the actomyosin filament [18]. Here, the second messenger cyclic AMP (cAMP) serves to " relax " endothelial cells by activating protein kinase A, which phosphorylates the GTPase RhoA, and thus inhibits generation of tension [19]; nonclassical relaxation mechanisms that bypass protein kinase A also exist [20] [21]. We have thus investigated to what extent elevation of cAMP levels could stabilize vessels in microfluidic scaffolds [16]. "
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    Microfluidic Cell Culture Systems, Edited by Christopher Bettinger, Jeffrey T. Borenstein, Sarah L. Tao, 12/2012: chapter 8: pages 205-221; Elsevier., ISBN: 978-1-4377-3459-1
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    • "The identity of the phosphatase remains unknown, although, a number of prominent phosphor-serine,-threonine protein phosphatases and a phosphotyrosine phosphatase are known to be activated by PKA in endothelial cells, including alkaline phosphatase and myosin light chain phosphatase. The latter plays an important role in cytoskeleton remodeling and the breakdown in vascular barrier permeability during disease (Aslam et al., 2010; Beuckmann et al., 1995; Goeckeler and Wysolmerski, 2005). Therefore, PKAdependent dephosphorylation of Mct1 may be a component of such pathological processes and it will be important to identify the phosphatase in future studies. "
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