ERK-Regulated Double Cortin-Like Kinase (DCLK)-Short Phosphorylation and Nuclear Translocation Stimulate POMC Gene Expression in Endocrine Melanotrope Cells

Abstract

We tested whether double cortin-like kinase-short (DCLK-short), a microtubule-associated Ser/Thr kinase predominantly expressed in the brain, is downstream of the ERK signaling pathway and is involved in proopiomelanocortin gene (POMC) expression in endocrine pituitary melanotrope cells of Xenopus laevis. Melanotropes form a well-established model to study physiological aspects of neuroendocrine plasticity. The amphibian X. laevis adapts its skin color to the background light intensity by the release of α-MSH from the melanotrope cell. In frogs on a white background, melanotropes are inactive but they are activated during adaptation to a black background. Our results show that melanotrope activation is associated with an increase in DCLK-short mRNA and with phosphorylation of DCLK-short at serine at position 30 (Ser-30). Upon cell activation phosphorylated Ser-30-DCLK-short was translocated from the cytoplasm into the nucleus, and the ERK blocker U0126 inhibited this process. The mutation of Ser-30 to alanine also inhibited the translocation and reduced POMC expression, whereas overexpression stimulated POMC expression. This is the first demonstration of DCLK-short in a native endocrine cell. We conclude that DCLK-short is physiologically regulated at both the level of its gene expression and protein phosphorylation and that the kinase is effectively regulating POMC gene expression upon its ERK-mediated phosphorylation.

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin

“…This finding is consistent with the observation that DCLK2 enters the nucleus after the cleavage of its C-terminus (a probable ‘regulatory domain’) ( Nagamine et al , 2014 ). Translocation from the cytoplasm to the nucleus has also been suggested for DCLK1 ( Burgess and Reiner, 2001 ; Kuribara et al , 2011 ). Autophosphorylation assays suggested that a DCLK3 protein with an apparent molecular weight close to that of the full-length form had the highest level of catalytic activity.…”

Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

“…In these studies we used U0126 to demonstrate a link between MEK1/2 cascade inhibition and DCLK1 expression in human pancreatic cancer cells and confirmed our results with microarray data generated by other groups in treated colon cancer cell lines (Figure  4D & Additional file 1: Figure S2F). Additionally, a previous paper demonstrated that nuclear translocation of phosphorylated DCLK1-short is attenuated in U0126-treated Xaenopus laevis melanotrope cells [46], which if broadly applicable, implies that MEK1/2 cascade inhibition has the potential to limit or prevent DCLK1-based intracellular signaling. The data from this study and our previous studies [7,8] suggest that DCLK1 interacts with and regulates the MAPK/ERK effector c-MYC.…”

“…A rabbit polyclonal antibody was commercially generated (Abbomax) to target ser-30(β)/336(α), which has previously been demonstrated to be phosphorylated, against a 15 amino acid peptide sequence similar to others previously used to generate phospho-DCLK1 antibodies [46,55]. To confirm specificity, ELISA was performed using phosphospecific and non-phosphospecific peptide.…”

DCLK1, a Putative Stem Cell Marker in Human Cholangiocarcinoma

“…Similar results were observed in the protein analysis by WB. In all populations, it was observed a 82kDa band corresponding to the most studied isoform from previous studies (23). Fig.1A show the expression of DCLK1 protein in pCCA-subpopulation compare to unsorted pCCA cells.…”

“…In all populations, a 82‐kDa band was observed, corresponding to the most studied isoform from previous studies. ( 23 ) Figure 1A shows the expression of DCLK1 protein in the pCCA subpopulation compared with unsorted pCCA cells. The pCCA LGR5+ (1.41 ± 0.2; n = 6; P < 0.01), pCCA CD90+ (0.68 ± 0.07; n = 6; P < 0.05), pCCA 133+ (0.74 ± 0.07; n = 6; P < 0.01), pCCA EpCAM+ (1.15 ± 0.19; n = 6; P < 0.01;), and pCCA CD13+ (1.3 ± 0.03; n = 6; P < 0.01) expressed higher protein levels compared with the unsorted control (0.45 ± 0.14; n = 6).…”