Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells

Abstract

Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (Akinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKAmediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.AKAP12 | melanoma | metastasis S caffold proteins, also known as anchoring proteins, are of crucial importance within cells because they allow dynamic association of signaling molecules, often present in low abundance, by concentrating them at discrete subcellular localizations, forming intricate signal transduction complexes. However, the mechanisms by which scaffold proteins are regulated within the tumor microenvironment and how altering their expression can result in a switch between kinase targets and promote tumor progression remain unknown. Importantly, scaffold proteins have recently been identified as new therapeutic targets, highlighting the necessity of understanding the biology behind these potential targets (1). In this paper, we focus on the scaffold protein AKAP12, which is a member of the larger "A Kinase Anchoring Protein" family that regulates signal transduction from the cell surface to the cytoskeleton. Previous studies have reported that AKAP12 binds to protein kinase A (PKA), protein kinase C (PKC), Src, and the actin cytoskeleton, all involved in tumorigenesis (2). However, the role of the tumor microenvironment in regulating the scaffolding protein AKAP12 and the outputs of these downstream signaling cascades has not been fully investigated.Hypoxia, or tumor cell oxygen deficiency, is a component of the tumor microenvironment and is strongly linked to metastasis and the poor clinical outcome of patients, through the activation of specific transcriptional, translational, and signaling programs (3-8). Induction of the hypoxia-inducible transcription factor (H...

FRET biosensors reveal AKAP-mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

“…Our findings that PKA activity was reduced in the cytosol may be an important consequence of gravin expression or upregulation in vivo . This possibility was recently highlighted in a study by Finger et al (2015), where hypoxia-induced gravin upregulation in human melanoma cells changed the phosphorylation profile of a wide variety of intracellular proteins [32]. Interestingly, the findings of Finger et al (2015) also support a second mechanistic observation from the current study, namely that gravin may direct the phosphorylation of a wider range of PKA substrates at or near the membrane than has been previously reported.…”

Retracted: AKAP12 mediates PKA-induced phosphorylation of ATR to enhance nucleotide excision repair

“…AKAP12 impacts tumor progression and metastasis through scaffolding of signaling molecules and downstream pathways that regulate tumorigenesis (30,31,63). AKAP12 expression is often downregulated in multiple cancer types, either associated with gene deletion, promoter hypermethylation or changes in chromatin modeling (64).…”

“…A-kinase anchoring proteins (AKAPs) are scaffolding proteins that regulate cellular cAMP responses by spatiotemporally coordinating PKA with target proteins specific to individual activation stimuli (27,28). AKAP12 (also called Gravin and SSeCKS) has been implicated in a wide range of cell functions, including tumor suppression (29–31), cytoskeletal architecture (32,33), β 2 -adrenergic receptor desensitization/resensitization (34,35) and cell cycle regulation (36–38). AKAP12 activities have been described at the plasma membrane, the cell periphery and at perinuclear regions of the cytoplasm (28).…”

The Different Routes to Metastasis via Hypoxia-Regulated Programs

“…AKAP12v2 is a direct HIF target and regulates protein kinase A (PKA) activity under hypoxia (2% O 2 for 16 h), favoring tumor cell invasion and migration in vitro , but most importantly, metastasis in an in vivo orthotopic model of melanoma [101]. Additionally, metastatic samples have higher levels of AKAP12v2 than the primary tumor has [101–105]. It is possible that hypoxia-induced splicing of AKAP12v2 at the primary site provides an advantage for dissemination and/or colonization at the metastatic sites.…”