Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Venkata Ramesh Dasari,Hindupur K Anandatheerthavarada,Marie-Anne Robin,Ettickan Boopathi,Gopa Biswas,Ji-Kang Fang,Daniel W Nebert,Narayan G Avadhani

doi:10.1074/jbc.m510725200

Venkata Ramesh Dasari, Hindupur K Anandatheerthavarada + Show 6 more

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https://doi.org/10.1074/jbc.m510725200

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Abstract

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.

Highlights

Recent studies from our laboratory showed that different xenobiotic-inducible CYPs such as rat CYP1A1, CYP2E1, and CYP2B1 and others that are widely recognized as microsomal proteins (MC-CYPs) are targeted to varying degrees to mitochondria (9 –11)
In the present study we have purified and characterized MT-CYP1A1 from livers of BNF-treated mice with three specific objectives: (a) to study the factors that regulate signal recognition particle (SRP) binding to nascent chains and proteolytic processing of CYP1A1 for bimodal targeting to the ER and MT; (b) to provide further supporting evidence for our hypothesis on the occurrence of chimeric signals that drive the bimodal protein targeting; and (c) to determine the evolutionary conservation of the endoproteolytic-processing mechanism for the activation of the cryptic MT-targeting signal in another animal besides the rat
Bimodal targeting of some of the ER (CYP1A1, CYP2B1, and CYP2E1) and plasma membrane proteins to mitochondria by virtue of their chimeric-targeting signals requires the activation of cryptic MT-targeting signals

Summary

Introduction

Recent studies from our laboratory showed that different xenobiotic-inducible CYPs such as rat CYP1A1, CYP2E1, and CYP2B1 and others that are widely recognized as microsomal proteins (MC-CYPs) are targeted to varying degrees to mitochondria (9 –11). These studies led to the concept of a new family of chimeric non-canonical-targeting signals, which function both as ER-targeting and MT-targeting signals, under different physiological conditions. Post-translational Processing of Mouse CYP1A1 Protein terminal 4th and 32nd amino acids of the protein by a cytosolic endoprotease, which exposes the MT-targeting signal, as reported for rat CYP1A1 [9], and (b) protein kinase A-mediated phosphorylation of the nascent chains at Ser-128 in CYP2B1 or Ser-129 in CYP2E1, which activates the cryptic MT-targeting signal. The results show that mouse MT-CYP1A1 actively metabolizes erythromycin, as well as a number of psychotropic drugs, including anti-depressants, anti-psychotics, and anesthetics in an FDX1 plus FDXR-supported system, exhibiting substrate specificity quite distinct from the full-length MC-CYP1A1 (ER-associated)

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