**3. Discussion**

Extensive e fforts have been focused in manipulating the activity of p38 MAPK for therapeutic purposes due to its close association with various pathological conditions, including inflammatory and malignant diseases [8]. Activation by phosphorylation via the upstream MKKs is the canonical mode of kinase activation. This study identifies, for the first time, a posttranslational modification of arginine methylation on Arg49 and Arg149 of p38 α by PRMT1 and demonstrates that methylation on R49/R149 modulates activation of p38 α through an increased association with the upstream MKK3 and the downstream e ffector MAPKAPK2 and thus impacts erythroid di fferentiation. This study elucidates a novel regulatory mechanism for p38 α activation and indicates a potential new strategy in intervening p38 α signaling.

Upon stimulation, a cascade of phosphorylation events involving MEKs and MKKs result in the phosphorylation of p38 MAPKs on the conserved Thr180-Gly-Tyr182 motif, leading to an open and extended activation loop, which allows for the binding of substrate and facilitates catalysis [21]. In addition to this canonical mode, increasing evidence has shown other modes of regulation. The phosphorylation of Thr180/Tyr182 of p38 can also be achieved by p38 auto-catalysis in response to T cell receptor (TCR)- or TNF α-mediated signaling in an MKK-independent manner [11,12]. The triggering of autophosphorylation can be induced by a precedent phosphorylation of p38 on Tyr323 by ZAP70, a T cell receptor-associated tyrosine kinase [11], or by the binding of TAB1, an adaptor protein in TNF α-mediated signaling, with p38 α [12]. In the MKK-dependent mode, docking regions on p38 mediate its interactions with various partners including MKKs, substrates, and phosphatases, in a context-dependent fashion, and contributes to the selective transduction of diverse signals [25]. GRK2 (G-protein-coupled-receptor kinase 2) phosphorylates p38, in a known docking groove, compromises the binding of MKK6 and thus suppresses the activation of p38 upon LPS stimulation in macrophages [10]. In this study, we show that arginine methylation of R49 and R149 by PRMT1 enhances the activation phosphorylation of p38 α upon induced erythroid di fferentiation (Figure 3C, Figure 4A,B). We identify that MKK3 is the responsible upstream kinase (Figure 5A,C,D and E). The enhanced activation of p38 α is mediated by an increased association with the upstream kinase MKK3 via R49/R149 methylation (Figure 5F,G). MKK6 is not an upstream kinase for p38 α in AraC-induced erythroid di fferentiation (Figure 5B,F) and is not associated with p38 α no matter whether R49 and R149 are wild-type or mutated (Figure 5H). These results uncover a novel mechanism for the regulation of p38 α signaling selectively through MKK3 by arginine methylation.

The correct recognition of MAPKs by the cognate interacting partners is critical for specifically transducing signals with a high fidelity. A common docking (CD) domain containing a few of conserved acidic residues is found in MAPKs that contribute mainly to the binding a ffinity [25,26]. The intervening sequences of these acidic residues and sequences in other regions play predominant roles in partner selectivity [25,26]. The sequence context for partner selection and binding a ffinity in response to di fferent stimuli is not fully understood. Arg49 and Arg149 are not located near the conserved acidic residues Asp313, Asp315, and Asp316 in the CD domain of p38 α. Arginine methylation catalyzed by PRMTs alters the hydrogen bonding capacity, hydrophobicity, and steric hindrance of the target arginine and its vicinity [27] and thus a ffects a wide range of protein properties. The activity of PRMT1 is up-regulated during erythroid di fferentiation [3], leading to the methylation of p38 α and the enhanced activation of the kinase (Figure 3C). Arginine methylation on Arg49/Arg149 can potentially cause a conformational change or an increased hydrophobicity favoring partner selection and/or binding a ffinity. In addition, the HRD sequence (His148-Arg149-Asp150) is shown to lock p38 α in an inactive conformation that can be disrupted by Tyr323 phosphorylation [28]. This observation raises the possibility that the methylation of R149 may facilitate a conformational change favorable for kinase activation. Arg49 is stereoscopically near Leu108 and Met109, which is part of a lipophilic pocket facilitating ATP binding [29]. The possibility that methylation on R49 favors the stabilization of this lipophilic pocket is worthy of further study.

p38 α is a versatile MAPK participating in many important physiological and pathological conditions [1–3]. The molecular events involved in its activation and regulation have attracted considerable research attention. A number of posttranslational modifications (PTMs) in p38 α have been identified via proteomics approaches [30]. However, relatively few of the identified PTMs are reported with functional impacts. In this study, we have identified several methylated arginine residues in p38 α. Among those, R49 and R149 are dimethylated only in the presence of PRMT1, suggesting they are PRMT1 substrate sites (Figure 1). Non-methylation mutants of R49K and R149K lost around 40% of methyl incorporation (Figure 1), indicating the existence of other potential PRMT1 sites. In cells, the activation phosphorylation of R49/149K mutant is remarkably reduced (Figure 4), which explains the incapability of the mutant to promote erythroid di fferentiation (Figure 2). The lack of Thr180-Gly-Tyr182 phosphorylation of the AGF mutant does not a ffect its arginine methylation level (Figure 3D), indicating that phosphorylation is not a prerequisite for methylation. Arginine methylation by PRMT1 significantly increases the association of p38 α with the upstream kinase MKK3 (Figures 5G and 6D) and the downstream substrate MAPKAPK2 (Figure 6C,D). This study reveals that the arginine methylation of p38 α on R49/R149 by PRMT1 renders the kinase more accessible to the enzyme (MKK3) and also facilitates its action toward the downstream e ffector MAPKAPK2. The importance of methylation in the MAPK signaling is also evidenced in other studies. The methylation of MAPK kinase kinase 2 (MAP3K2) on Lys260 by SMYD3 (SET and MYND domain containing 3) blocks its interaction with the negative regulator PP2A phosphatase and activate Ras-mediated MEK/ERK (extracellular signal-regulated kinase) signaling [31]. The arginine methylation of Raf by PRMT5 results in an increased degradation, a decreased Ras-Raf-Erk signaling and a reduced proliferation of PC12 cells upon EGF (epidermal growth factor) stimulation [32]. Our results demonstrate that, in addition to erythroid di fferentiation, the sorbitol-stimulated activation of p38 α is also enhanced by PRMT1 and dependent on the methylation of R49 and R149 (Figure 4C,D). Together, methylation, in collaboration with phosphorylation, is emerging as a critical regulatory mechanism of the MAPK pathways via which extracellular cues are integrated to elicit an appropriate response in terms of signal magnitude, duration and specificity.

Although p38 α has been shown to be a critical player in various stages during erythroid di fferentiation, its upstream activating kinase and downstream substrate had not been clearly and fully revealed. Our results decisively show that only MKK3, not MKK6, mediates the activation of p38 α in AraC-induced erythroid di fferentiation (Figure 5A,B). The immediate downstream e ffectors of p38 α in erythroid di fferentiation are much less described. We identify that MAPKAPK2 participates in erythroid di fferentiation via p38 α signaling (Figure 6). The RNA-binding protein human antigen R (HuR) protein is an e ffector of MAPKAPK2 that can bind and stabilize GATA1 transcripts during embryonic erythropoiesis [33]. GATA1 is a critical transcription factor upregulated during erythroid di fferentiation [34] in a p38 α-dependent fashion (Figure 2C). Whether MAPKAK2 promotes erythroid di fferentiation by stabilizing GATA1 transcripts via HuR is of interest.

Genetically modified mice have evidenced that the p38 α pathway plays important roles in inflammatory responses and hematopoietic homeostasis among others [8]. A number of pharmacological inhibitors of p38 MAPK have been developed and tested in clinical trials for treating inflammatory diseases, such as rheumatoid arthritis and chronic obstructive pulmonary disease and hematopoietic diseases, such as myelodysplastic syndromes (MDS), which frequently leads to hematological malignancy [35]. Major drawbacks for p38 inhibitors are the isotype specificity and the undesired side e ffects often observed with kinase inhibitors. This study unveils a novel regulatory mechanism for p38 α activation and signaling through arginine methylation and provides a new strategy, other than kinase inhibition, in intervening p38 α signaling.

#### **4. Materials and Methods**

#### *4.1. Materials and Plasmid*

1-beta-D-arabinofuranosylcytosine (AraC) and benzidine were obtained from Sigma-Aldrich. S-adenosyl-L-[methyl-3H] methionine (3H-AdoMet, 0.55 mCi/ml, NET-155H) and fluorographic enhancer, EN3HANCE, were from PerkinElmer. The pFlag-CMV2-p38 α plasmid [3] was used as a template for PCR to generate mutations on R49 and R149. These plasmids were subsequently cloned into the pET6H vector for expression of recombinant proteins.

## *4.2. Cell Culture*

The human chronic myelogenous leukemia (CML) K562 cells were purchased from BCRC (Bioresource Collection and Research Center, Taiwan) and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 IU/mL streptomycin and 100 IU/mL penicillin as described. The pLKO.1 puro-based shRNAs, including MKK3-sh1, MKK6-sh1, MKK6-sh2, MAPKAPK2-sh1, MAPKAPK2-sh2, were purchased from National RNAi Core Facility, Taiwan. The transfection of K562 was performed by using LipofectamineTM 2000 Reagent (Invitrogen). Stable clones of gene knockdown were selected in the presence of puromycin (0.5 μg/mL). The p38 α-knockdown cell clones were generated by the same procedure as described [3,4]. The protein levels of p38 α in these knockdown cells were around 20% of the parental cells.

#### *4.3. Expression and Purification of Recombinant Proteins*

Recombinant His-tagged p38 α WT and mutants (R49K, R149K and R49/149K) proteins were expressed in *E. coli* BL21 (DE3) pLysS by isopropyl-beta-D-thiogalactoside (IPTG) (0.4 mM) induction and immobilized on Ni + -NTA agarose (Qiagen). Proteins were eluted with elution bu ffer (20 mM Tris-HCl pH7.9, 0.5 M NaCl, and 0.6 M imidazole) and dialyzed to remove imidazole. The purified proteins were stored in a bu ffer containing 20 mM Tris-HCl pH7.9 and 50 mM NaCl. The recombinant GST-fused PRMT1 proteins were expressed in *E. coli* BL21 by IPTG (0.2 mM) induction and immobilized on glutathione Sepharose 4B (GE Healthcare). Proteins were eluted with and stored in bu ffer (50 mM Tris-HCl Ph 7.9 and 20 mM reduced glutathione).
