Phosphorylation of proteins on serine/threonine residues is often initiated by or coupled with tyrosine phosphoryla-tion. In fact, tyrosine phosphorylation has so far been considered the primary or even the exclusive indicator of signal transduction in multicellular organisms. Enhanced PTK activity has been implicated in not only cancer and proliferative diseases, but also in inflammatory disorders. Inhibition of PTKs effectively blocks the increase in endothelial permeability induced by a wide spectrum of inflammatory mediators, whereas upregulation of tyrosine phosphorylation with tyrosine phosphatase inhibitors exerts opposite effects.
Protein tyrosine kinases can be categorized into receptor tyrosine kinases, such as the VEGF receptor KDR, and nonreceptor tyrosine kinases, such as Src kinases and focal adhesion kinase (FAK). The Src family, containing c-Src, Lyn, Fyn, Lck, Hck, Fgr, Blk, and Yes, exerts potent phos-phorylating and transforming effects through interaction with the SH2 and SH3 domains of effector proteins. The activity of Src is upregulated by phosphorylation at Tyr-416 (located in the catalytic domain) and negatively regulated by phosphorylation at Tyr-527 (near the carboxyl terminus). It has been documented that the Src signaling is required in multiple cellular processes, such as cell contraction and migration, angiogenesis, and vascular leakage. Blockage of Src activity or Src deficiency reduces cerebral edema associated with stroke in animal models. Recent experiments show that Src mediates vascular endothelial permeability responses to tumor necrosis factor and reactive oxygen metabolites. In addition, specific blockage of Src activity by pharmacological agents or Src-inhibiting peptides abolishes the increase in albumin permeability caused by C5a-activated neutrophils in isolated venules as well as in cultured venular endothelial monolayers. Further biochemical analysis confirms that activated neutrophils stimulate Src phosphorylation at Tyr-416 and decrease phosphorylation at Tyr-527, two events that are known to upregulate Src activity.
Several pathways may be activated through Src signaling. For example, MAPK has been identified as a downstream effector of Src. More importantly, Src may directly alter endothelial barrier structure by phosphorylating contractile proteins and adhesion proteins. Supporting this is biochemical evidence of coimmunoprecipitation and immunocolocalization between Src and the cytoskeletal and junctional components. Myosin light chain kinase, b-catenin, and FAK have been identified as potential Src substrates. Therefore, it is highly possible that Src activation serves as a common signal in coordinating cytoskeletal contraction, junctional disorganization, and focal adhesion redistribution in response to multiple protein kinase cascades that are triggered by inflammatory mediators.
Another nonreceptor tyrosine kinase known to modulate endothelial barrier structure and function is FAK. Focal adhesions are referred to as transmembrane structures that anchor the cells to extracellular matrices in the basement membrane. Major components of the focal structure include a family of transmembrane receptors, namely integrins, and their associated intracellular proteins, such as talin, a-actinin, and paxillin, which link the cytoskeleton to inte-grins. Not only does this structure support the physical attachment of endothelial lining to the basement membrane, it also serves as a transducer in mediating cellular response to physical stress or chemical stimuli. Because of the lack of catalytic activity in integrin receptors, such signaling reactions are transduced via a network of integrin-associated proteins located in focal adhesions. Of these proteins, FAK is a major protein kinase capable of catalyzing various downstream signaling cascades leading to a dynamic regulation of focal adhesion morphology and distribution. The activity of FAK is mainly regulated through tyrosine phosphorylation, preferentially by the Src-family tyrosine kinases. FAK-mediated focal adhesion assembly and reorganization play a critical role in cell contraction and migration during stimulation by integrins or nonintegrin factors including shear stress, growth factors, and permeability-increasing agents such as thrombin, platelet activating factors, phorbol esters, and even leukocyte adhesion molecules. Recent evidence is accumulating in support of the functional importance of FAK to the microvascular barrier effect. For example, there are reports that protein tyrosine kinase blockades abolish FAK phosphorylation-coupled endothelial barrier dysfunction. Downregulation of FAK expression using antisense approaches modulates agonist-induced increases in endothelial monolayer permeability. More importantly, inhibition of FAK activity through direct delivery of FAK-related nonkinase (FRNK), an endogenously expressed FAK competitive inhibitor, into intact isolated venules significantly attenuates the hyperpermeability response to VEGF or activated leukocytes.
Several pathways may be considered for potentially underlying the effect of FAK on endothelial barrier function. On one hand, FAK-signaled focal adhesion disassembly and redistribution may reduce cell-matrix adhesion forces, leading to rounding of the endothelial cell and widening of interendothelial channels. This is consistent with an emerging view that FAK activation leads to disassembly, rather than assembly, of focal adhesion complexes. On the other hand, activated FAK may directly participate in the development of contractile force in the endothelial cells resulting in intercellular gap formation. In support of this notion, FAK phosphorylation has been linked to actin polymerization and stress fiber formation in endothelial cells treated with VEGF. Alternatively, FAK could indirectly affect endothe-lial barrier function by coordinating secondary intracellular signaling molecules, including the Src family of tyrosine kinases and the Rho family of GTPases. In this regard, FAK may function as a scaffolding protein that recruits and activates Src, Rho, and other focal adhesion-associated proteins, which in turn cause changes in the cyto-skeleton or intercellular junctions, leading to endothelial hyperpermeability.
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