In conclusion high methylation levels of CDKL were observed

In conclusion, high methylation levels of CDKL2 were observed both in HCC cell lines and tissues, which showed a negative association with mRNA expression. Moreover, treatment with demethylation agent in HCC cell lines could upregulate CDKL2 mRNA expression. The discovery of the epigenetic modification of CDKL2 may be a new horizon for HCC investigation and promote the exploration of novel potential methylation biomarkers for HCC. The following are the supplementary data related to this article.
Funding This work was supported by the Science and Technology Research Plan of Wuhan City (2015060101010057), the Fundamental Research Funds for the Central Universities (2042017kf0087).
Introduction Cyclin-dependent kinase 5 (CDK5) is one of the proline-directed serine/threonine kinases but with functions distinct from other CDKs (Nakano et al., 2005). CDK5 is a small serine/threonine kinase that is abundant in post-mitotic neurons (Zhao et al., 2009). The activity of CDKs is tightly regulated by an intricate system of protein–protein interactions and phosphorylations (Morgan, 1995). Activation of CDK5 requires an association with p35 (Patrick et al., 1998), which has been shown to be a rate-limiting factor for CDK5 activity (Takahashi et al., 2005). Also, it has been reported that CDK7, which is involved in transcriptional regulation (Larochelle et al., 1998), functions as a CDK5 activating kinase in Cyclosporin H (Rosales et al., 2003). We established a CDK5-knockdown mouse model, which showed a distinct phenotype of a change in hair color compared to the wild type (Dong et al., 2017). Analysis of the transcriptome profile of the skin of CDK5-knockdown mice further showed that the mitogen-activated protein kinase (MAPK) pathway was one of the pathways affected by the loss of CDK5, which included Mitogen-activated protein kinase kinase kinase 6 (MAP3K6) (Supplementary Table 1). MAPK signaling pathways regulate almost all cellular processes such as proliferation, differentiation, apoptosis, and survival through interconnected signaling cascades (Cho et al., 2006). In melanocytes, the melanins including eumelanins and pheomelanins are produced and transferred around keratinocytes, during which melanogenesis is stimulated by the master transcriptional regulator microphthalmia-associated transcription factor (MITF) via activating some pigmentation genes, including tyrosinase (TYR), tyrosinase related protein-1 (TYRP1), dopachrome-tautomerase (DCT), premelanosome protein (PMEL), and melan-A (MLANA) (Yasumoto et al., 1994, Bertolotto et al., 1998, Du et al., 2003). The components of the MAPK signaling pathway phosphorylate Ser73 in the MITF protein, resulting in its ubiquitination and subsequent degradation (Hemesath et al., 1998). It has been reported that the MAPK/extracellular regulated protein kinase (ERK) pathway may contribute to the inhibition of melanin production (Chae et al., 2017). Activated MAPK and ribosomal S kinases (RSKs) are translocated to the nucleus and may modify several transcription factors, notably cAMP-responsive element-binding protein (CREB), which mediates MITF-M transcription (Yu et al., 2003, Price et al., 1998). MAP3K6 was originally identified as a member of the serine/threonine protein kinase family owing to its interaction with MAP3K5/ASK1 and a protein kinase that also activates the transcription factor c-Jun and p38 kinase (Wang et al., 1998), which is specifically expressed in tissues that are directly exposed to the external environment of the body, such as the skin, lungs, and gastrointestinal tract (Iriyama et al., 2009, Takeda et al., 2007). MAP3K6, known as apoptosis signal-regulating kinase 2 (ASK2), is a member of the MAPK signaling cascade (Kyriakis and Avruch, 2012) and a kinase involved in MAPK activation (Hemesath et al., 1998). Taken together, we put forward to a hypothesis that CDK5 might play roles in melanogenesis through MAPK signaling pathway. Here, we evaluated the effect of CDK5 on regulation of the MAPK pathway in the skin of CDK5-knockdown mice to further supply the underlying mechanism of the functional role of CDK5 in melanogenesis.