RPS6KA3/RSK2-mediated phosphorylation of DRAM2 promotes lysosomal targeting and autophagic flux in melanoma

Macroautophagy/autophagy is a critical process for maintaining cellular homeostasis and has emerging implications in cancer biology. DRAM2 (DNA damage regulated autophagy modulator 2), a transmembrane protein enriched at lysosomal membranes, has been implicated in autophagy regulation; however, the upstream mechanisms governing its trafficking and function remain unclear. In this study, we identified RPS6KA3/RSK2, a stress-responsive kinase downstream of the MAPK pathway, as a novel upstream kinase of DRAM2. RPS6KA3/RSK2 interacted with and phosphorylated DRAM2 at Ser263 within its cytosolic tail. This phosphorylation was required for AP3D1/AP-3-dependent trafficking of DRAM2 to the late endosomal-lysosomal pathway, thereby facilitating autolysosome formation and sustaining autophagic flux. In contrast, the non-phosphorylatable DRAM2^S263A mutant failed to bind AP3D1/AP-3, exhibited defective lysosomal trafficking, and was partially redistributed toward plasma membrane-proximal compartments, where it enhanced exosome secretion. Bioinformatic analyses revealed a strong positive correlation between RPS6KA3/RSK2 and DRAM2 expression in melanoma tissues, and elevated DRAM2 expression was associated with poor patient prognosis. Depletion of RPS6KA3/RSK2 or DRAM2 impaired autophagic flux and inhibited melanoma cell proliferation. Similarly, expression of the DRAM2^S263A mutant suppressed melanoma progression in vitro and in vivo by disrupting autophagy. Moreover, DRAM2 protein levels were elevated in skin cancer tissues compared to normal tissues. Collectively, our findings uncover a phosphorylation-dependent trafficking switch that bifurcates DRAM2 function between autophagy and exosome secretion, and establish the RPS6KA3/RSK2-DRAM2 axis as a critical regulator of melanoma progression. This signaling pathway may represent a promising therapeutic target for autophagy-associated malignancies.Abbreviations: AGC: protein kinase A, G, and C families; AP-3: adaptor protein 3; CD: cytosolic domain; CSNK2/CK2: casein kinase 2; co-IP: co-immunoprecipitation; CQ: chloroquine; CREB: cAMP responsive element binding protein; CTKD: C-terminal kinase domain; DRAM2: DNA damage regulated autophagy modulator 2; EBSS: Earle's balanced salt solution; ESCRT: endosomal sorting complexes required for transport; GEPIA: gene expression profiling interactive analysis; GPS: global positioning system; GRK7: G protein-coupled receptor kinase 7; GSK3B: glycogen synthase kinase 3 beta; IP-MS: immunoprecipitation-mass spectrometry; LAMP1: lysosome associated membrane protein 1; LAMP2: lysosome associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK: mitogen-activated protein kinase; MVB: multivesicular body; NTA: nanoparticle tracking analysis; NTKD: N-terminal kinase domain; PI4K2: phosphatidylinositol 4-kinase type 2 alpha; PRKAA2: protein kinase AMP-activated catalytic subunit alpha 2; RPS6KA3/RSK2: ribosomal protein S6 kinase A3; SKCM: skin cutaneous melanoma; SQSTM1: sequestosome 1.