Cross-species Sequence Analysis Reveals Multiple Charged Residue-rich Domains That Regulate Nuclear/Cytoplasmic Partitioning and Membrane Localization of A Kinase Anchoring Protein 12 (SSeCKS/Gravin)

Jeffrey W Streb,Joseph M Miano

doi:10.1074/jbc.m414017200

Abstract

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.

Highlights

§ To whom correspondence should be addressed: Center for Cardiovascular Research, Aab Inst. of Biomedical Sciences, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642
An N-terminal myristoylation motif targets the AKAP12␣ protein to the endoplasmic reticulum, mutants lacking this motif localize in a similar manner as the ␤ and ␥ isoforms
Given that targeting motifs regulate a key property of A kinase anchoring proteins (AKAPs) function and are likely to be well conserved across species, we hypothesized that these basic regions may be important determinants of AKAP12 localization

Summary

Introduction

§ To whom correspondence should be addressed: Center for Cardiovascular Research, Aab Inst. of Biomedical Sciences, University of Rochester School of Medicine, 601 Elmwood Ave., Rochester, NY 14642. Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Whereas deletion of the C-terminal most 404 amino acids does not affect localization, further truncation of the C terminus (⌬791–1607) results in redistribution of AKAP12-GFP chimeras to the nucleus (Fig. 1D).

Results

Conclusion