Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting

ABSTRACT Mitogen-activated protein (MAP) kinases are stable enzymes that are mainly regulated by phosphorylation and subcellular targeting. Here we report that extracellular signal-regulated kinase 3 (ERK3), unlike other MAP kinases, is an unstable protein that is constitutively degraded in proliferating cells with a half-life of 30 min. The proteolysis of ERK3 is executed by the proteasome and requires ubiquitination of the protein. Contrary to other protein kinases, the catalytic activity of ERK3 is not responsible for its short half-life. Instead, analysis of ERK1/ERK3 chimeras revealed the presence of two destabilization regions (NDR1 and -2) in the N-terminal lobe of the ERK3 kinase domain that are both necessary and sufficient to target ERK3 and heterologous proteins for proteasomal degradation. To assess the physiological relevance of the rapid turnover of ERK3, we monitored the expression of the kinase in different cellular models of differentiation. We observed that ERK3 markedly accumulates during differentiation of PC12 and C2C12 cells into the neuronal and muscle lineage, respectively. The accumulation of ERK3 during myogenic differentiation is associated with the time-dependent stabilization of the protein. Terminal skeletal muscle differentiation is accompanied by cell cycle withdrawal. Interestingly, we found that expression of stabilized forms of ERK3 causes G1 arrest in NIH 3T3 cells. We propose that ERK3 biological activity is regulated by its cellular abundance through the control of protein stability.

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Localization of the rat myosin I molecules myr 1 and myr 2 and in vivo targeting of their tail domains

Journal of Cell Science ◽

10.1242/jcs.108.12.3775 ◽

1995 ◽

Vol 108 (12) ◽

pp. 3775-3786 ◽

Cited By ~ 9

Author(s):

C. Ruppert ◽

J. Godel ◽

R.T. Muller ◽

R. Kroschewski ◽

J. Reinhard ◽

...

Keyword(s):

Subcellular Localization ◽

Brush Border ◽

Subcellular Fractionation ◽

Actin Binding ◽

Resting Cells ◽

Medium Density ◽

Subcellular Targeting ◽

Myosin I ◽

Punctate Pattern

Myr 1 is a widely distributed mammalian myosin I molecule related to brush border myosin 1. A second widely distributed myosin I molecule similar to myr 1 and brush border myosin I, called myr 2, has now been identified. Specific antibodies and expression of epitope-tagged molecules were used to determine the subcellular localization of myr 1 and myr 2 in NRK cells. Myr 1 was detected at the plasma membrane and was particularly enriched in cell protrusions like lamellipodia, membrane ruffles and filopodia. In dividing cells myr 1 localized to the cleavage furrow. Myr 2 was localized in a discrete punctate pattern in resting cells and in cells undergoing cytokinesis. In subcellular fractionation experiments myr 1 and myr 2 were both partly soluble and partly associated with smooth membranes of medium density. The tail domains of myosin I molecules have been proposed to interact with a receptor and thereby determine the subcellular localization. To test this hypothesis we expressed the tail domains of myr 1 and myr 2 that lack the F-actin-binding myosin head domain in NRK cells. These tail domains also partly copurified with smooth membranes of medium density and immunolocalized similar to the respective endogenous myosin I; however, they exhibited a lower affinity for membranes and an increased diffuse cytosolic localization. These results suggest that the tail domains of myr 1 and myr 2 are sufficient for subcellular targeting but that their head domains also contribute significantly to maintaining a proper subcellular localization.

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Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816614115 ◽

2018 ◽

Vol 115 (49) ◽

pp. E11465-E11474 ◽

Cited By ~ 15

Author(s):

F. Donelson Smith ◽

Mitchell H. Omar ◽

Patrick J. Nygren ◽

Joseph Soughayer ◽

Naoto Hoshi ◽

...

Keyword(s):

Solid Phase ◽

Second Messenger ◽

Nuclear Accumulation ◽

Nucleotide Polymorphisms ◽

Binding Assays ◽

Subcellular Targeting ◽

Regulatory Subunits ◽

Anchoring Proteins ◽

Anchoring Protein ◽

Second Messenger Signaling

A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

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A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

BMC Cell Biology ◽

10.1186/1471-2121-10-26 ◽

2009 ◽

Vol 10 (1) ◽

pp. 26 ◽

Cited By ~ 11

Author(s):

Nicholas F Garrone ◽

Bonnie L Blazer-Yost ◽

Robert B Weiss ◽

Jean-Marc Lalouel ◽

Andreas Rohrwasser

Keyword(s):

C2 Domain ◽

Subcellular Targeting ◽

Human Polymorphism

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Identification of Novel Plant Peroxisomal Targeting Signals by a Combination of Machine Learning Methods and in Vivo Subcellular Targeting Analyses

The Plant Cell ◽

10.1105/tpc.111.084095 ◽

2011 ◽

Vol 23 (4) ◽

pp. 1556-1572 ◽

Cited By ~ 74

Author(s):

Thomas Lingner ◽

Amr R. Kataya ◽

Gerardo E. Antonicelli ◽

Aline Benichou ◽

Kjersti Nilssen ◽

...

Keyword(s):

Machine Learning ◽

Subcellular Targeting ◽

Learning Methods ◽

Machine Learning Methods ◽

Peroxisomal Targeting ◽

Targeting Signals

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Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

Molecular and Cellular Biology ◽

10.1128/mcb.26.8.2947-2954.2006 ◽

2006 ◽

Vol 26 (8) ◽

pp. 2947-2954 ◽

Cited By ~ 38

Author(s):

Wei Gong ◽

Michael Russell ◽

Keiko Suzuki ◽

Karl Riabowol

Keyword(s):

Kinase Inhibitor ◽

Biological Activities ◽

Ectopic Expression ◽

Growth Stress ◽

Nuclear Localization Sequence ◽

Binding Motif ◽

Subcellular Targeting ◽

Cyclin Dependent Kinase Inhibitor ◽

Cargo Proteins ◽

Localization Sequence

ABSTRACT ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33ING1b increased levels of the p21Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33ING1b, consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization.

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