**1. Introduction**

The p38 mitogen-activated protein kinases (MAPKs) play pivotal roles in a number of cellular processes, such as inflammation, stress response, differentiation, and survival [1]. The four members (p38<sup>α</sup>, p38β, p38γ and p38δ) of the family share high sequence homology and common regulatory mechanisms. However, they also exhibit characteristic biochemical properties and unique cellular functions [2]. The dysregulation of p38α activity plays a dominant role in various pathological conditions. The in vitro and in vivo experiments demonstrate an essential role of p38α in various stages of hematopoiesis, including erythroid differentiation, megakaryocytic differentiation, and myelopoiesis and the involvement in hematological diseases such as myelodysplastic syndromes [3–6]. The concerted regulation of p38α with other signaling pathways can either promote tumorigenesis or suppress tumor progression as shown in colon and breast cancer, respectively [7,8]. p38α triggers the production of inflammatory mediators and cytokines, such as IL-1βand IL-6, upon stimulation [8,9]. The dysregulated activity is linked to a variety of inflammatory diseases, including chronic obstructive pulmonary disease (COPD), colitis, and rheumatoid arthritis [8,9]. The intervention of p38α signaling with an aim toward disease treatment has attracted considerable attention.

p38 α MAPK is activated through a canonical cascade involving phosphorylation by upstream dual-specificity MAPK kinases (MKKs), particularly MKK3 and MKK6, on the sequence-specific threonine and tyrosine residues (Thr180 and Tyr182) located in the activation loop. This phosphorylation confers a conformational change that allows the binding of substrates and the accessibility of the catalytic center. Dual-specificity phosphatases responsible for de-phosphorylating Thr180/Tyr182 control the magnitude and duration of the signaling [2]. Accumulating evidence demonstrates the existence of alternative (or additional) mechanisms that regulate p38 activation. GRK2 (G-protein-coupled receptor kinase 2) phosphorylates p38 α on Thr123 located at the docking groove for MKKs, which impairs the binding of MKK6 to p38 α and diminishes the activation of p38 upon LPS (lipopolysaccharide) stimulation [10]. ZAP70 (zetachain-associated protein kinase 70) and TAB1 (transforming growth factor β-activated protein kinase 1 (TAK1) binding protein 1) can activate p38 α in T cell receptor (TCR)-mediated signaling and myocardial injury, respectively [11,12]. ZAP70 phosphorylates p38 α on Tyr 323, leading to autophosphorylation on Thr180 and the activation of the kinase [11]. TAB1 interacts with p38 α to trigger a conformational change, leading to the autophosphorylation of p38 on Thr180 [12]. Acetylation of Lys53 in the ATP-binding pocket of p38 α by PCAF/p300 increases its a ffinity for ATP and thus enhances the kinase activity during hypertrophy of cardiomyocytes [13]. Moreover, protein arginine methyltransferase 1 (PRMT1) promotes the activation of p38 α during erythroid di fferentiation [3]. However, the detailed molecular mechanism is ye<sup>t</sup> to be revealed. A full understanding of the regulatory mechanisms of p38 α activity will provide alternative ways to develop strategies which di fferentiate p38 α functions from other isozymes for the therapeutic needs.

PRMT1 is the predominant protein arginine methyltransferase regulating various cellular processes, including gene transcription, DNA repair, and signal transduction [14]. PRMT1 catalyzes the addition of mono- or di-methyl groups to arginine residues, leading to the alteration of protein/protein interaction, protein/nucleic acid interaction, enzymatic activity and other posttranslational modifications [15]. The malfunction of PRMT1 is tightly associated with many pathological conditions, including hematological malignancy [16,17]. PRMT1 plays important roles in hematopoiesis. PRMT1 is required for adult erythroid and lymphocyte di fferentiation, as shown by a conditional *Prmt1* knockout mouse model [18]. PRMT1 can methylate RUNX1, a transcription factor critical for hematopoiesis, resulting in the impairment of its association with co-repressor SIN3A, and thus impacts the maturation of myeloid and erythroid lineages in cell models [17]. PRMT1 promotes erythroid di fferentiation by enhancing the activation of p38 α in response to EPO (erythropoietin) and AraC (1-beta-arabinofuranosyl) induction in hematopoietic CD34+ progenitor and K562 cells, respectively [3]. Ca2+ influx is an essential event in various stages during erythroid di fferentiation [19,20]. We have shown that Ca2+ up-regulates the activity of PRMT1 and stimulates erythroid di fferentiation via the novel Ca2+-PRMT1-p38 α axis [20].

In this study, we identified Arg49 and Arg149 of p38 α as PRMT1 methylation sites by in vitro methylation followed by mass spectrometric analysis (liquid chromatography-tandem mass spectrometry, LC-MS/MS). The non-methylation mutations of Lys49/Lys149 and Ala49/Ala149 abolished the promotive e ffect of p38 α on erythroid di fferentiation. The activation phosphorylation of R49/149K mutant p38 α was greatly reduced upon induced di fferentiation. The interaction of mutant p38 α with the upstream MKK3 was significantly reduced. These results indicate that arginine methylation on R49/R149 enhances the interaction of p38 α with MKK3 and thus promotes activation phosphorylation by MKK3. This study also indicates that phosphorylation by MKK3 is not a prerequisite for methylation by PRMT1 and PRMT1 acts directly on p38 α. In addition, we identified that MAPKAPK2 (MAPK-activated protein kinase 2) was a p38 α downstream e ffector involved in PRMT1-mediated promotion of erythroid di fferentiation. Interaction of MAPKAPK2 with p38 α was also significantly reduced in the R49/149K mutant. Together, this study unveils a novel regulatory mechanism of p38 α activation via protein arginine methylation on R49 and R149 by PRMT1, which impacts the interaction of p38 α with its upstream kinase MKK3 and downstream substrate MAPKAPK2 and thus promotes erythroid di fferentiation.
