Supplemental material for Myosin light chain kinase (<i>MYLK</i>) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

<p>Supplemental material for Myosin light chain kinase (<i>MYLK</i>) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions by Ting Wang, Mary E. Brown, Gabriel T. Kelly, Sara M. Camp, Joseph B. Mascarenhas, Xiaoguang Sun, Steven M. Dudek and Joe G.N. Garcia in Pulmonary Circulation</p

Structure–Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of <i>MYLK</i> Variants

<div><p>The <i>MYLK</i> gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three <i>MYLK</i> SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by ¹H-¹⁵N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by <i>in silico</i> molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these <i>MYLK</i> SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm <i>MYLK</i> as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.</p></div

Selection of the N-terminal segments of nmMLCK1.

<p>The segment of 1-494aa was initially selected for protein expression within the N-terminal sequence of nmMLCK1, containing three ALI-associated SNPs and two phosphorylatable Tyr sites, Y464 and Y471. This sequence generates a protein of ca. 53 kDa, within a suitable range of size for practical bacterial protein expression and survived preliminary NMR trials. Included in this ca. 500aa protein are three immunoglobulin C-2 type (IGc2) domains and a low-complexity region (preceding the 3rd IGc2 domain) as predicted by SMART. Subsequently, a shorter 1-264aa segment of ca. 28 kDa was also generated spanning the three ALI-associated SNPs (two IGc2 domains) and exhibited advantages for NMR-based structural determination.</p

Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta

<div><p>Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue–specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.</p></div

Comparison of the HSQC spectra of ¹⁵N-labeled 1-264aa/ 1-494aa segments of nmMLCK1.

<p>(A) and (B), the HSQC spectra of 1-264aa segments of nmMLCK1: (A) wild type; (B) P147 SNP variant. (C) to (F), the superimposition of the HSQC spectra (the 1st set of spectra shown in <i>blue</i> color and the 2nd in <i>red</i> color): (C) wild type, 1-494aa and 1-264aa; (D) P147, 1-494aa and 1-264aa; (E) 1-264aa, wild type and 147P; (F) zoom in of the squared region in (E). The HSQC spectra of the 1-264aa segments exhibited a better dispersion of ^<b>1</b>H-^<b>15</b>N chemical shifts, with less degenerate, better resolved signals than 1-494aa segments. Superimposition of the spectra of the 1-264aa segments onto those of their corresponding 1-494aa segments demonstrated that the spectra of 1-264aa segments are a recapitulation of the subsets of those of their corresponding 1-494aa segments, suggesting that the shorter segments each possess a structure similar to the corresponding part of their longer counterparts. The same characteristic pattern of signal changes observed for 1-494aa segments is recapitulated with better resolution by superimposition of the spectra of the 1-264aa 147P SNP mutant onto those of the 1-264aa wild type segment, suggesting that the same structural difference exist for the 1-264aa wild type and the P147 SNP mutant.</p

Potential involvement of S147P and P21H SNP sites in nmMLCK1 in phosphorylation-dependent binding of 14-3-3 proteins.

<p>(A) Sequence alignment of human nmMLCK1 (wild type and SNPs) with the consensus of 14-3-3 binding modes and selected protein kinase substrates as well as its murine ortholog. The “S” or “T” in <i>red</i> indicates a phophorylation site, with underline indicating a predicted site (<i>black</i> underlined) or a confirmed site (<i>red</i> underlined). The “R” or “K” in <i>blue</i> and the “P” in <i>green</i> indicate their potential involvement in key binding recognition. The “<u>P</u>” indicates a “P” that may be missing and hence nonessential. The “X” in <i>black</i> indicates any amino acid. The “<u>X</u>” indicates an “X” that may be missing and hence nonessential. The consensus of GSK3 substrate (<u>S</u>XXX<u>S</u>, the 2nd <u>S</u> representing a pre-existing pSer or pThr) is shown in repeat in order to align with multiple potential phosphorylation sites in nmMLCK1. While not shown, additional multiple alignments of S147, S18, and other nearby Ser residues including S16, S26, S145 and S154, are possible with the highly variable consensus substrate R<u>XX</u>X<u>S</u> of the AGC group of protein kinases that include PKA, PKG and PKC families, suggesting a complex regulation of nmMLCK via these SNP-embracing loops by different kinase-mediated phosphorylation and subsequent binding to 14-3-3 proteins. (B) Molecular modeling of the 1-252aa segment of nmMLCK1 [template: deleted in colorectal cancer (DCC) (PDBID: 3lafA)] revealing localization of S147P and P21H SNP sites in separate loops at the two ends of the single, 1st IGc2 domain (with side chains of some loop residues of interest shown), despite that the modeling of loop conformations may be of poor quality. (C) Immunoprecipitation (IP) of Flag-tagged nmMLCK1 wild type using Flag-M5 anitibody followed by western blot using pan-14-3-3 antibody indicating the binding of 14-3-3 proteins to nmMLCK1 before and after S1P stimulation. Note: In the IP result shown, the bottom 14-3-3 bands correspond to 24 kDa and the top 14-3-3 bands correspond to 27 kDa.</p

Comparison of the HSQC spectra of ¹⁵N-labeled 1-494aa segments of nmMLCK1.

<p>(A) Wild type and P147; (B) wild type and H21-P147; (C) wild type and H21-P147-A261; (D) wild type and A261; (E) H21-P147 and H21-P147-A261; (F) H21-P147 and P147. By superimposing the spectra of the SNP variants (the 2nd set of spectra, in <i>red</i> color) onto wild type or other SNP variants (the 1st set of spectra, in <i>blue</i> color), we have identified characteristic changes in the HSQC signals corresponding to each individual SNP mutation versus the wild type, which are indicated along red (S147P), pink (P21H) and brown (V261A) lines, respectively (see insets for more details). The characteristic patterns associated with these three SNPs suggest that these SNP mutations are indeed distant to each other in the tertiary structure and therefore cause only minor or minimal local conformational changes and independent changes in the HSQC signals.</p

Proposed roles of PKCδ in thrombin-induced human lung EC barrier disruption through a pathway involving MLC and Rho GTPase.

<p>Thrombin activates PKCδ that then leads to the activation of PKD and CPI-17. In turn, PKD activates the RhoA pathway inducing MLC activation, while PKCδ phosphorylates CPI-17, a potent MLCP inhibitor, thereby resulting in increased MLC phosphorylation. MLC phosphorylation mediates actin stress fiber formation and contraction. In addition, PKCδ inhibits Rac1 activity blocking its barrier protective effects upon thrombin stimulation.</p

Effect of rottlerin pretreatment on thrombin-induced PKD and CPI-17 phosphorylation.

<p><b>(A)</b> EC were pretreated with rottlerin (10 μM, 30 min) followed by stimulation with thrombin (1U/ml, 5 min) and whole cell lysates were immunoblotted with anti-phospho-PKD and anti-PKD antibody. Unstimulated cells (Un) were used as controls. <b>(B)</b> Normalized densitometry analysis of PKD phosphorylation is shown (n ≥ 3/condition, * p < 0.05). <b>(C)</b> In subsequent experiments using the same conditions, whole cell lysates were immunoblotted with anti-phospho-CPI-17 and anti-CPI-17 antibody. <b>(D)</b> Normalized densitometry of CPI-17 phosphorylation is shown. n ≥ 3/condition, * p < 0.05.</p

Effect of PKCδ inhibition on thrombin-induced MLC phosphorylation.

<p>(<b>A)</b> EC were pretreated with rottlerin (5–10 μM, 30 min) and subsequently stimulated with thrombin (1U/ml, 5 min). Whole cell lysates were immunoblotted with anti-phospho-MLC (Thr18/Ser19) antibody. (<b>B)</b> Relative MLC phosphorylation by densitometry is shown (n ≥ 3/condition, * p < 0.05).</p