The actin cytoskeleton is an important regulator of various cellular functions in ECs including proliferation, migration and permeability 
. To gain insights into the molecular processes underlying HLA class I-mediated actin stress fiber remodeling we conducted a proteomic analysis of the cytoskeletal fractions of ECs treated with anti-HLA class I antibodies and contrasted these findings to thrombin and bFGF-induced cytoskeletal remodeling. Here we report a novel method to examine the composition of the EC cytoskeleton and reveal unique cytoskeleton proteomes in ECs depending on the agonist used for stimulation. Using annotation tools, a candidate list was created that revealed 12 potential HLA class I induced targets that may play a role in understanding EC cytoskeleton changes.
Among the list of candidates, we identified proteins or related proteins involved in cytoskeleton dynamics. For example, cortactin is a known actin-binding protein and transduces signaling to the cytoskeleton 
. It also has the ability to cross-link F-actin, which is regulated by Src-kinase mediated activation 
. Additionally, the actin-related protein 2/3 (Arp2/3) complex is essential in the regulation of actin polymerization and localizes to sites of dynamic actin 
. To tie these processes together one group reported that cortactin colocalizes with both dynamic actin and the Arp2/3 complex 
. HLA class I ligation on the surface of ECs stimulates the formation of actin stress fibers and the activation of Src family kinases 
. Thus, it is plausible that HLA class I-mediated Src kinase phosphorylation may regulate cortactin binding protein 2 and Arp 2/3 complex subunit 3 in the formation of stress fibers following class I ligation.
In the past, we established that HLA class I signaling cascades involve various phosphorylation events and have explored several key proteins responsible for these events 
. The current data add to our understanding of these phosphorylation events as we explored the kinases that have the potential to phosphorylate the candidate proteins. Among the kinase predictions, cyclin dependent kinase 2 (CDK2 has the potential to phosphorylate 3 of the candidate proteins. CDK2 has already been established as a regulator of HLA class I signal transduction. HLA class I ligation on the surface of ECs leads to the inactivation of retinoblastoma protein (Rb) and this happens through CDK2 
. Rb is negative regulator of cell cycle progression 
. Activation of CDK2 relieves the inhibition put forth by Rb and this permits the ECs to pass the G1
checkpoint and proliferate 
. The current study elucidated several candidate targets of CDK2, which may contribute to a better understanding of the molecular processes involved in HLA class I-mediated signal transduction leading to cell proliferation.
Another kinase family predicted to be involved in the phosphorylation of the candidate proteins is 70-kDa S6 protein kinase (p70S6k) family, where the specific kinase was ribosomal protein S6 kinase 1 (S6RP). HLA class I ligation leads to the phosphorylation of p70S6k and S6RP, which are downstream of mTOR complex 1 
. It has yet to be determined which S6RP downstream targets are important in HLA class I signaling. The current findings suggest that S6RP may phosphorylate two of the candidate proteins, ATP-dependent RNA helicase DDX3X and nuclear pore complex protein Nup153. DDX3X was confirmed by Western blot to be associated with the isolated cytoskeleton fraction following HLA class I ligation on ECs. DDX3X interacts with TANK binding kinase 1 to activate the interferon (IFN) promoter. When DDX3X protein levels are reduced using RNAi, type I IFN production is diminished 
. Cytokine production is associated with allograft rejection 
and in vitro
, cytokines are known to synergize with HLA class I antibodies to enhance signal transduction 
. Little is known about the regulation of HLA class I induced cytokine production and DDX3X may be a potential target in controlling cytokine induction. Recent findings suggest that Nup153 is crucial for nucleoskeleton and cytoskeleton architecture maintenance and is necessary for cell cycle progression and cell migration. In addition, when Nup 153 is knocked down by RNAi, there is prominent cytoskeleton rearrangement that hinders cell migration in human breast carcinoma cells 
. The integrity of the cytoskeleton is key to HLA class I signaling 
and given the role of Nup153 it seems that this protein would be worth investigating.
TPM4 was identified as a candidate protein in the HLA class I treated group by mass spectrometry and confirmed by Western blot to be present only in the cytoskeleton fraction of HLA class I stimulated ECs. Tropomyosins are among the most abundant cytoskeletal proteins in ECs 
. By mass spectrometry, tropomyosin-1 was identified as an oxidative-stress-sensitive phosphoprotein in ECs 
. H2O2 induced the activation of DAP kinase, downstream of ERK. DAP kinase promoted the phosphorylation of tropomyosin-1, which was essential for H2O2 induced formation of actin stress fibers in ECs 
. HLA class I ligation leads to ERK activation 
and thus, it would be valuable to determine if TPM4 functions downstream of ERK as regulator of HLA class I induced cytoskeleton remodeling.
The eIF4A1 protein, functions downstream of mTOR complex 1, which has been shown to phosphorylate 4E-BP1 following class I ligation 
. 4E-BP1 is bound to eIF4E and when 4E-BP1 becomes phosphorylated eIF4E is released and recruited to eIF4F, which includes both eIF4A1 and eIF4G. This complex promotes translation and cell proliferation 
. The link between translational machinery and the cytoskeleton was postulated some time ago 
. Continued efforts to make this connection showed that when the soluble fraction of a cell lysate was compared to the cytoskeleton fraction, certain initiation factors where enriched in the cytoskeleton and one of those factors was eIF4A1 
. It has yet to be determined how the cytoskeleton could regulate and organize translation.
Although eIF4A1 and DDX3X were found by mass spectrometry to be exclusively in the HLA class I treated EC, surprisingly these proteins were found at approximately equal levels in all of the treatment groups following cytoskeleton isolation and Western blotting. The most likely explanation for the detection of eIF4A1 and DDX3X by mass spectrometry only in the class I treated group is the difference in the sensitivity of these assays. In the ECs treated with bFGF or thrombin, other more abundant cytoskeletal proteins may have been present which reduced the relative amount of DDX3X and eIF4A1, precluding their detection by mass spectrometry. Indeed, even when highly sensitive mass spectrometers are used to analyze complex biological samples and bodily fluids, high-abundance proteins obscure the detection of lower-abundance proteins 
An alternative explanation for these discrepant findings is that different protein:protein interactions in the treatment groups influence the ability to detect a protein by mass spectrometry. For example, the function of eIF4A1 has little to do with its protein level and more to do with whether or not it is being inhibited 
. Programmed Cell Death 4 (PDCD4) is a tumor suppressor known to bind eIF4A1, which inhibits translation initiation and proliferation 
. Detection of eIF4A1 in the cytoskeleton of all treatment groups by Western blot would not be hindered if it is bound to PDCD4, but detection by mass spectrometry could be masked.
We found that treatment with HLA class I antibodies or thrombin stimulated varying degrees of colocalization between eIF4A1 and F-actin and paxillin suggesting that eIF4A1 may interact with specific compartments of the cytoskeleton in a unique manner. Consistent with this concept, recent studies by our laboratory identified two different signaling pathways leading to MLC phosphorylation and stress fiber formation in ECs, depending upon the nature of the stimulus (ME Ziegler, unpublished). Stimulation with thrombin at 1 U/ml induced a robust increase in the intracellular Ca2+
concentration, increased phosphorylation of MLC and promoted stress fiber formation via MLCK and ROK in an ERK-independent manner. In contrast, stimulation of ECs with a low dose of thrombin (1 mU/ml) or HLA class I antibodies did not promote any detectable change in intracellular Ca2+
concentration, but induced MLC phosphorylation and stress fiber assembly via MLCK and ROK in an ERK1/2-dependent manner. HLA class I ligation requires the recruitment of integrin ß4 in order to activate proliferation and migration 
. In carcinoma migration, α6ß4 integrin functions via its ability to promote and stabilize F-actin 
. Additionally, eukaryotic initiation factors in migrating cells localize with focal adhesions, which contain actin and this up-regulation of localized translation is thought to be a result of integrin engagement 
. These data suggest that the increased colocalization of eIF4A1 with F-actin and paxillin following class I ligation may be in response to the HLA class I molecule partnering with integrin ß4 to elicit intracellular signals. Although the colocalization data supports a molecular interaction between eIF4A1 and F-actin and paxillin, additional experiments are required to definitively prove a physical interaction between these proteins. eIF4A1 was suggested to be a potential target for developing new anti-cancer and anti-inflammatory drugs given evidence of cross-talk between translation and the inflammatory response 
. Our findings suggest that eIF4A1 may also be a potential therapeutic target of HLA class I induced antibody-mediated rejection.
An important functional consequence of HLA class I ligation on ECs is stimulation of cell proliferation, which we previously reported to occur in subconfluent ECs 
. To gain knowledge on the cytoskeleton changes involved in class I-mediated cell proliferation, we used subconfluent ECs to conduct these experiments. However, the EC density is an important factor that influences cellular responses. Confluent ECs regulate thrombosis, inflammation, vascular cell proliferation and matrix remodeling, whereas subconfluent ECs promote these events 
. Cell morphology, expression of cell surface molecules and behavior of the ECs differ in subconfluent versus confluent ECs 
. Additionally, the EC response to an agonist varies depending on cell density 
. A quiescent EC monolayer is more similar to the in vivo
setting in appearance and differentiated properties 
. We have yet to explore the cytoskeletal proteome of a confluent monolayer of ECs in response to HLA class I ligation and postulate that a confluent monolayer may respond differently and utilize distinct signaling pathways.
A key question is how signal transduction is orchestrated through these molecular interactions to stimulate actin cytoskeletal remodeling. Our previous publications and current data are consistent with a model whereby molecular aggregation of HLA class I molecules with antibodies leads to recruitment of integrin ß4 and the subsequent activation of intracellular signals that increase Rho-GTP activity, induce phosphorylation of ROK and trigger the assembly and phosphorylation of FAK, Src and paxillin at the focal adhesions to stimulate actin reorganization 
. The new candidate proteins identified in this study may further contribute to this model. Candidates such as TPM4, eIF4A1, DDX3X, cortactin binding protein 2 and Arp2/3 may be recruited to the focal adhesions to regulate cell proliferation and survival. Similar signaling cascades have been described following antibody cross-linking of ICAM-1 on ECs. ICAM-1 ligation induced cytoskeleton changes, which included increased intracellular calcium, protein kinase C activation, phosphorylation of cortactin and other actin-binding proteins by Src, activation of RhoA GTPase, and subsequent rearrangements of the actin 
In conclusion, these studies provide new information that can be applied to the exploration of known pathways. Given that phosphoprotein signal transduction is essential to HLA class I EC activation, not only are the proteins relevant, but also their corresponding kinases. Thus, validation of these proteins and examination of their activation state will be important in future studies. Overall these studies may reveal more specific targets in understanding the mechanisms of HLA class I induced antibody-mediated rejection. Additionally, these methods can be applied to other cell types and agonists as an effort to understand the role of cytoskeleton changes in many pathways.