Alexander D. Verin

Serine/threonine protein phosphatases in the control of cell function

Reversible protein phosphorylation is a fundamental mechanism by which many biological functions are regulated. Achievement of such control requires the coordinated action of the interconverting enzymes, the protein kinases and protein phosphatases. By comparison with protein kinases, a limited number of protein phosphatase catalytic subunits are present in the cell, which raises the question of how such a small number of dephosphorylating enzymes can counterbalance the action of the more numerous protein kinases. In mammalian cells, four major classes of Ser/Thr-specific phosphatase catalytic subunits have been identified, comprising two distinct gene families. The high degree of homology among members of the same family, PP1, PP2A and PP2B, and the high degree of evolutionary conservation between organisms as divergent as mammals and yeast, implies that these enzymes are involved in fundamental cell functions. Type 1 enzymes appear to acquire specificity by association with targeting regulatory subunits which direct the enzymes to specific cellular compartments, confer substrate specificity and control enzyme activity. In spite of the progress made in determining the structure of the PP2A subunits, very little is known about the control of this activity and about substrate selection. Recent studies have unravelled a significant number of regulatory subunits. The potential existence of five distinct B or B-related polypeptides, some of which are present in multiple isoforms, two A and two C subunit isoforms, raises the possibility that a combinatorial association could generate a large number of specific PP2A forms with different substrate specificity and/or cellular localization. Moreover, biochemical, biological and genetic studies all concur in suggesting that the regulatory subunits may play an important role in determining the properties of the Ser/Thr protein phosphatases and hence their physiological functions.

Regulation of Endothelial Barrier Function by the cAMP-Dependent Protein Kinase

Elevation of cAMP promotes the endothelial cell (EC) barrier and protects the lung from edema development. Thus, we tested the hypothesis that both increases and decreases in PKA modulate EC function and coordinate distribution of regulatory, adherence, and cytoskeletal proteins. Inhibition of PKA activity by RpcAMPS and activation by cholera toxin was verified by assay of kemptide phosphorylation in digitonin permeabilized EC. Inhibition of PKA by RpcAMPS or overexpression of the endogenous inhibitor, PKI, decreased monolayer electrical impedance and exacerbated the decreases produced by agonists (thrombin and PMA). RpcAMPS directly increased F-actin content and organization into stress fibers, increased co-staining of actin with both phosphatase 2B and myosin light chain kinase (MLCK), caused reorganization of focal adhesions, and decreased catenin at cell borders. These findings are similar to those evoked by thrombin. In contrast, cholera toxin prevented the agonist-induced resistance decrease and protein redistribution. Although PKA activation attenuated thrombin-induced myosin light chain (MLC) phosphorylation, PKA inhibition per se did not cause MLC phosphorylation or affect [Ca2+]i. These studies indicate that a decrease in PKA activity alone can produce disruption of barrier function via mechanisms not involving MLCK and support a central role for cAMP/PKA in regulation of cytoskeletal and adhesive protein function in EC which correlates with altered barrier function.

Role of Tyrosine Phosphorylation in Thrombin-Induced Endothelial Cell Contraction and Barrier Function

Thrombin-induced endothelial cell (EC) barrier dysfunction is highly dependent upon phosphorylation of serine and threonine residues present on myosin light chains (MLC) catalyzed by a novel EC myosin light chain kinase (MLCK) isoform. In this study, we examined the participation of tyrosine protein phosphorylation in EC contraction, gap formation and barrier dysfunction. We first determined that thrombin significantly increases protein tyrosine kinase activity and protein tyrosine phosphorylation in bovine pulmonary artery EC. Tyrosine kinase inhibitors, genistein and 2,5 DHC, reduced EC tyrosine kinase activities, however, only genistein significantly attenuated thrombin-mediated increases in albumin clearance and reductions in transendothelial electrical resistance. Similarly, genistein but not 2,5 DHC, decreased basal and thrombin-induced Ca2+ increases and MLC phosphorylation in the absence of alterations in Type 1 or 2A serine/threonine phosphatase activities. Immunoprecipitation of the EC MLCK isoform revealed a 214 kD immunoreactive phosphotyrosine protein and genistein pretreatment significantly reduced MLCK activity in MLCK immunoprecipitates. Although thrombin induced the translocation of p60src from the cytosol to the EC cytoskeleton, a detectable increase in the level of MLCK tyrosine phosphorylation was not noted after thrombin challenge. Taken together, our data suggest that genistein-sensitive tyrosine kinase activities are involved in thrombin-mediated EC MLCK activation, MLC phosphorylation, and barrier dysfunction.

Regulation of endothelial cell myosin light chain phosphorylation and permeability by vanadate

The involvement of tyrosine protein phosphorylation in the regulation of endothelial cell (EC) contraction and barrier function is poorly understood. We have previously shown that myosin light chain (MLC) phosphorylation catalyzed by a novel 214 kDa EC myosin light chain kinase (MLCK) isoform is a key event in EC contraction and barrier dysfunction [Garcia et al. (1995): J Cell Physiol 163:510–522; Garcia et al. (1997): Am J Respir Cell Mol Biol 16:487–491]. In this study, we tested the hypothesis that tyrosine phosphatases participate in the regulation of EC contraction and barrier function via modulation of MLCK activity. The tyrosine phosphatase inhibitor, sodium orthovanadate (vanadate), significantly decreased electrical resistance across bovine EC monolayers and increased albumin permeability consistent with EC barrier impairment. Vanadate significantly increased EC MLC phosphorylation in a time‐dependent manner (maximal increase observed at 10 min) and augmented both the MLC phosphorylation and permeability responses produced by thrombin, an agonist which rapidly increases tyrosine kinase activities. The vanadate‐mediated increase in MLC phosphorylation was not associated with alterations in either phosphorylase A Ser/Thr phosphatase activities or in cytosolic [Ca2+] but was strongly associated with significant increases in EC MLCK phosphotyrosine content. These data suggest that tyrosine phosphatase activities may participate in EC contractile and barrier responses via the regulation of the tyrosine phosphorylation status of EC MLCK. J. Cell. Biochem. 70:141–155, 1998.

Regulation of interleukin-1-stimulated GMCSF mRNA levels in human endothelium

The regulation of interleukin-1 (IL-1)-mediated increases in GMCSF mRNA levels in human endothelium was examined and determined to occur in a time- and protein kinase C (PKC)-dependent manner. IL-1beta induced the early activation and translocation of PKC isotypes alpha and beta2 to the nucleus and PKC inhibition attenuated the IL-1-mediated increase in GMCSF mRNA levels. PKC activation by PMA alone, in the absence of IL-1beta activation, however, was insufficient to allow GMCSF mRNA detection. Increasing cyclic adenosine nucleotide (cAMP) levels suppressed IL-1beta-induced increases in GMCSF mRNA levels. In contrast, botulinum toxin C, which mediates the ADP ribosylation of a 21 kD ras-related G protein, augmented IL-1beta-induced GMCSF mRNA expression. Inhibition of protein synthesis (with cycloheximide) raised basal GMCSF mRNA transcripts to detectable levels, augmented IL-1-induced increases in GMCSF mRNA levels, and exhibited negative regulation by cAMP. Finally, disruption of either microtubules (with colchicine) or microfilaments (with cytochalasin B) resulted in reduced GMCSF mRNA expression in response to IL-1beta. These results are compatible with a model wherein IL-1-mediated increases in human endothelial cell GMCSF mRNA may be linked to both nuclear protein kinase C activation and activation of a low molecular weight G-protein, although neither activity alone is sufficient to increase the levels of GMCSF mRNA.