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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Immunol. Author manuscript; available in PMC Mar 1, 2011.
Published in final edited form as:
PMCID: PMC2832913
NIHMSID: NIHMS164765

Interleukin-1 Receptor-associated Kinase-1 (IRAK-1) functionally Associates with PKCε and VASP in the Regulation of Macrophage Migration

Abstract

Macrophage migration is mediated by complex cellular signaling processes and cytoskeleton re-arrangement. In particular, recent advances indicate that the innate immunity signaling process plays a key role in the regulation of macrophage migration. In this report, we have provided evidence demonstrating the involvement of a key innate immunity signaling kinase, Interleukin-1 Receptor-Associated Kinase-1(IRAK-1) as a critical modulator of macrophage migration. Macrophage migration induced by phorbol 12-myristate 13-acetate (PMA) is significantly attenuated in IRAK-1−/− macrophages as compared to wild type macrophages. Mechanistically, we demonstrated that IRAK-1 works downstream of PKCε and upstream of VASP, a member of Ena/VASP family proteins. IRAK-1 forms a close complex with PKCε as well as VASP, and participates in PMA-induced phosphorylation of VASP. Notably, IRAK-1 contains a novel EVH1 domain binding motif (L167WPPPP) within its N-terminus, which is responsible for its interaction with VASP. The mutant IRAK-1 (L167A/W168A) fails to associate with VASP. Our findings provide a novel facet regarding the molecular signaling process regulating macrophage migration.

INTRODUCTION

Macrophage migration is regulated at multiple levels by diverse molecules including cellular receptors, intracellular signaling proteins, and cytoskeletal structural molecules[13]. The regulated expressions of cytokines, chemokines, as well as chemokine receptors during the immune cell activation and migration have been extensively studied in the past[4, 5]. However, relatively little information is available regarding the regulation of structural proteins by innate immunity signaling processes.

VASP (VAsodilator-Stimulated Phosphoprotein) is one of the key structural proteins involved in the regulation of cell migration because it is responsible for facilitating cellular focal adhesion, as well as actin filament binding and polymerization [6]. VASP contains a novel EVH1 domain at its N-terminus that binds with protein partners containing a rare L (W/F) PPPPS/T motif [7]. Almost all of the currently identified proteins containing such a motif are structural proteins including zyxin and vanculin [8]. However, no regulatory proteins (e.g. protein kinases or phosphatases) have been identified that bind directly with VASP.

The Interleukin-1 Receptor Associate Kinase 1 (IRAK-1) is a critical protein kinase involved in innate immunity signaling processes[911]. IRAK-1 is known to be not only involved in the inducible expression of pro-inflammatory mediators [12, 13], but also in macrophage migration. IRAK-1 deficient mice have decreased monocyte/macrophage infiltration to inflamed brain tissues in the EAE model[14]. However, the molecular mechanism underlying IRAK-1 mediated macrophage migration is not clearly understood. Intriguingly, we noticed that IRAK-1 contains a perfect match of the LWPPPPSP motif within the highly Pro-Ser rich region, raising the potential prospect that IRAK-1 may serve as a direct regulatory kinase involved in VASP phosphorylation and function. Intriguingly, phosphorylation of VASP at Serine 157 has been well identified to be necessary for agonist-induced cell migration[15, 16]. In particular, PMA-mediated activation of protein kinase C (PKC) is known to contribute to VASP hosphorylation at Ser157 [17, 18]. Recent study also revealed that IRAK-1 is associated and regulated by various isoforms of PKCs[19, 20].

In this report, we tested the hypothesis that IRAK-1 may be a close interacting partner with VASP. Furthermore, we tested the functional relationship between IRAK-1, PKC, and VASP phosphorylation, as well as its implication in macrophage migration.

MATERIALS AND METHODS

Mice and cells

Wild type (WT) C57BL/6 mice were purchased from the Charles River laboratory. IRAK-1−/− mice on a C57BL/6 background were kindly provided by Dr. James Thomas from the University of Texas Southwestern Medical School. All mice were housed and bred at Derring Hall animal facility in compliance with approved Animal Care and Use Committee protocols at Virginia Polytechnic Institute and State University. THP-1 cells, an undifferentiated human pro-monocytic cell line, were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Bone marrow derived macrophages (BMDMs) were isolated from WT and IRAK-1−/− mice and maintained as described [21]. MAT-2 cells, obtained from Dr. Fabio Re, were Hela cells stably transfected with TLR2 and were maintained as described[21].

Plasmids and transfection

The wild type pFlag-IRAK-1 plasmid, and the C-terminal deletion pFlag-IRAK-1ΔC plasmids were made as described[21]. The pFlag-IRAK1-L167A/W168A was generated using the GeneEditor site-directed mutagenesis kit (Promega, WI). All constructs were confirmed by automated sequencing. Plasmids were transiently transfected into MAT-2 cells using the lipofectamine 2000 as described by the manufacturer (Invitrogen).

Co-immunoprecipitation and western blot

Cells were washed in phosphate-buffered saline and subsequently lysed with lysis buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA, 20 mM β-glycerophosphate, 1 mM NaF, 5mM p-nitrophenylphosphate, 1mM Na3VO4, 100×protease inhibitors). 800µg of cell extract was incubated for 1h with either anti-IRAK-1, anti-VASP (Santa Cruz) or control IgG (Santa Cruz) antibodies followed by overnight incubation with 60µl of Protein A/G Plus agarose (Sigma). The samples were separated on 10% SDS-polyacrylamide gels and then Western blotted with anti-PKCε (Cell signaling), anti-IRAK-1 or anti-VASP antibodies. Horseradish peroxidase conjugated secondary antibody was then added and the blot was developed employing ECL (Amersham Biosciences).

Trans-well cell migration assays

Trans-well migration assays were performed using 8-µm-pore-size trans-well inserts according to the manufacturer's instructions (BD Biosciences). Briefly, BMDMs (4× 105) suspended in serum free DMEM medium were added to the top chambers of trans-well inserts in 24-well plate. Stimulators were added to the lower chamber which contains DMEM/1% FBS. Cells were incubated for a proper amount of time, fixed, and then stained with Giemsa solution (Sigma). The numbers of migrated cells present were counted under microscope and expressed as means ± standard deviations.

Statistical analysis

Statistical significance was determined using the unpaired 2-tailed Student’s t-test and P-values less than 0.05 were considered statistically significant.

RESULTS

IRAK-1 physically associates with VASP in vivo

Given the presence of a novel EVH1-binding LWPPPPSP motif within IRAK-1, we tested whether IRAK-1 may form a close complex with VASP. Human monocytic THP1- cells were used to perform co-immunoprecipitation studies using anti-VASP antibody. As shown in Fig. 1A, IRAK-1 was detected in the VASP immuno-complex. To further confirm their interaction, we performed immunoprecipitation using an anti-IRAK-1 antibody. As shown in figure 1B, VASP was detected in the IRAK-1 co-immunoprecipitates in THP-1 cells. As confirmatory evidence, we performed immunofluorecence staining analyses of murine macrophages, and detected sub-cellular co-localization of IRAK-1 and VASP near the periphery of macrophages (data not shown).

Fig.1
IRAK-1 interacts with VASP. (A) Human monocytic THP1- cells were either untreated or treated with 40nM PMA for 15 min. Equal amounts of total cell lysates were harvested and used to perform immunoprecipitation analyses using an anti-VASP antibody. Co-immunoprecipitated ...

The LWPPPP motif within IRAK-1 is required for its interaction with VASP

To better define the structural requirement for their interaction, we have generated various IRAK-1 mutant plasmids. Specifically, we have designed plasmids encoding the full length IRAK-1 (pFlag-IRAK-1), IRAK-1 with C-terminal truncation (pFlag-IRAK-1ΔC), or IRAK-1 containing a point mutation at the LWPPPP motif, pFlag-IRAK-1(L167A/W168A) (Fig. 2B). These plasmids were transiently transfected into MAT-2 cells, and all transfected constructs were similarly expressed, as confirmed by Western blot (Fig. 2A). Equal amounts of cell lysates from transfected cells were used to perform co-immunoprecipitation assays using anti-VASP antibody. As shown in Fig.2A, pFlag-IRAK-1 and pFlag-IRAK-1-ΔC co-immunoprecipitated with VASP. By marked contrast, the mutant pFlag-IRAK-1(L167A/W168A) was not found in the VASP co-immunoprecipitates. These results suggest that VASP binding may be mediated by the LWPPPP site located between the death domain and the kinase domain of IRAK-1.

Fig.2
Identification of the IRAK-1 motif is necessary for its interaction with VASP. (A) MAT-2 cells were transiently transfected with either pFlag-IRAK-1, pFlag-IRAK-1ΔC, or pFlag-IRAK-1(L167A/W168A) mutants. Equal amounts of lysate were harvested ...

IRAK-1 and PKCε facilitates PMA-induced VASP phosphorylation at Ser157

Since IRAK-1 has been shown to associate with PKC, and PKC has been implicated in VASP phosphorylation at serine 157, we subsequently tested whether IRAK-1 is involved in PMA-induced VASP phosphorylation. BMDMs from WT or IRAK-1 deficient mice were treated with PMA for various time periods, and the phosphorylation status of VASP was evaluated by Western blot. As shown in Figure 3, PMA treatment induced a rapid occurrence of an upper phosphorylated VASP band in WT BMDMs. In contrast, PMA-induced VASP phosphorylation was significantly reduced in IRAK-1−/− BMDMs.

Fig.3
IRAK-1 is involved in PMA-induced VASP phosphorylation. (A) BMDMs from WT and IRAK1−/− BMDM mice were treated with 40nM PMA for the indicated time periods. Equal amounts of total cell lysates were resolved on SDS-PAGE and blotted with ...

We further examined the VASP phosphorylation using a VASP-pSer157 antibody. As shown in Fig. 3, there was a rapid induction of VASP phosphorylation at serine 157 in WT BMDMs. In contrast, there was a ~70% reduction of serine 157 phosphorylation in IRAK-1−/− BMDMs.

We also tested the interaction between IRAK-1 and PKCε, a known PKC isoform involved in the phosphorylation of VASP and cell migration[22]. As shown in Fig. 4A, IRAK-1 was co-precipitated with the endogenous PKCε in WT BMDMs.

Fig.4
IRAK-1 interacts with PKCε. (A) BMDMs from WT mice were either untreated or treated with 40nM PMA for 15 min. Equal amounts of total cell lysates were harvested and used to perform immunoprecipitation analyses using an anti-IRAK-1 antibody. Co-immunoprecipitated ...

To confirm that PMA-induced VASP phosphorylation was dependent on PKCε activation, a PKCε–specific inhibitor, myristoylated PKCεV1-2 peptide (BIOMOL), was utilized. As shown in Fig. 4B, PMA-induced VASP phosphorylation was completely abrogated by the addition of the inhibitor peptide.

Taken together, these results indicate that both IRAK-1 and PKCε functionally associate with each other in mediating PMA-induced VASP phosphorylation.

IRAK-1 participates in PMA-induced trans-swell migration of macrophages

To test whether IRAK-1 is required for PMA-induced macrophage migration, we performed trans-well migration assays using WT and IRAK-1−/− BMDMs. As shown in Figure 5, PMA treatment induced robust migration of WT BMDMs especially at 40nM. In contrast, there was a 70% reduction in cell migration with IRAK-1−/− BMDMs.

Fig.5
Depletion of IRAK-1 impairs cell migration. BMDMs from WT and IRAK1−/− mice were loaded onto the top wells of the trans-well chamber without or with PMA added to the bottom chamber at concentration of 4nM, 40nM or 400nM. After incubation ...

DISCUSSION

In this study, we demonstrated that IRAK-1 is a novel regulatory kinase closely associated with VASP and responsible for macrophage migration. First, we documented that IRAK-1 interacts with VASP via the LWPPPP motif within IRAK-1. Second, IRAK-1 coordinates with PKC and participates in PMA-induced VASP phosphorylation. Third, IRAK-1 is involved in PMA-induced macrophage migration.

The LWPPPPSP motif is located within the variable region, a region which is uniquely present in IRAK-1 molecule, as it is absent in all other forms of the IRAK family members (IRAK-2, M, and 4). All family members of IRAK-1 share a conserved N-terminal death domain and a kinase domain[23]. In sharp contrast, there is a novel variable region between the death domain and the kinase domain present only in IRAK-1 molecule. Despite the known presence of this domain, no biochemical or physiological function has been assigned to this portion of IRAK-1 molecule. Our study showed a requirement for this novel motif in the interaction of IRAK-1 with a cytoplasm structural protein, VASP. However, the molecular mechanism underlying the requirement for their interaction still requires further study. The LWPPPP motif may either directly participate in the interaction with VASP, or indirectly contribute via altering the secondary structure of other related domains involved in their interaction.

Functionally, our study reveals a glimpse of the dynamic and complex interplay between PKCε, IRAK-1 and VASP phosphorylation. PMA is the potent and specific activator of PKC. Given our finding that PMA-induced VASP phosphorylation is significantly attenuated in IRAK-1−/− cells, we reason that IRAK-1 works downstream of PKCε, and contributes to the phosphorylation of VASP. On the other hand, blocking of PKCε with a selective inhibitor completely ablates PMA-induced VASP phosphorlylation. This indicates that PKC may also directly contribute to VASP phosphorylation. Alternatively, IRAK-1 may serve as a positive feedback regulator, further inducing the activation of PKCε. Consistent with this scenario, a recent report indicates that TLR agonists can induce the activation of PKCε through a MyD88-p38 dependent pathway[24]. IRAK-1 is known an activator downstream of MyD88 and is upstream of p38 activation. Conceivably, IRAK-1 activation may lead to p38 activation and PKC phosphorylation and activation. In either of the above two scenarios, deletion of IRAK-1 would attenuate, but not completely ablate, PMA-induced activation of PKC and VASP phosphorylation. Although our data clearly demonstrate the involvement of IRAK-1 in the phosphorylation of VASP, We can not exclude the possibility that IRAK-1 may indirectly contribute to the phosphorylation of VASP. For example, PKC or other related and un-identified enzymes may be recruited to the complex due to IRAK-1, and responsible for VASP phosphorylation.

Our study reveals the pleiotropic nature of IRAK-1 function. IRAK-1 is known to be involved in cellular signaling networks controlling the activation of multiple transcription factors (e.g. NFκB[25], IRF5/7[2628], NFAT[29], and nuclear receptor transcription factors [30, 31]). In addition, IRAK-1 is present in both cellular cytoplasm and nucleus [32]. How IRAK-1 is capable of performing diverse functions is not clearly understood. Conceivably, the multiple functional domains of IRAK-1, including its kinase domain, an N-terminal death domain, a variable region containing the LWPPPP motif, and a C-terminal Serine-rich region, may help explain its participation in diverse cellular functions. Further biochemical and physiological studies are needed to clarify this question.

Acknowledgment

This work is partially supported by funding from National Institute of Health.

Footnotes

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