Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase

Hartigan, Jennifer A.; Green, E.G.; Mortensen, Richard M.; Menachery, Alphonsa; Williams, Gordon H.; Orme-Johnson, N R

doi:10.1016/0960-0760(95)00026-v

Cited by 30 publications

(18 citation statements)

References 26 publications

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“…5C). These observations are in agreement with earlier reports from the laboratories of Orme-Johnson and Stocco (2,(12)(13)(14). In contrast to wild-type StAR, the C-His-tag StAR was not proteolytically processed in the COS-1 cells and the C-His-tag StAR that was associated with the organelles was digested by proteinase K (Fig.…”

Section: His-tag Star Proteinssupporting

confidence: 83%

“…Thus, the minimally effective concentration of StAR needed to stimulate mitochondrial pregnenolone synthesis in vivo cannot be estimated. We recently presented evidence that phosphorylation of StAR at serine residue 195 by protein kinase A is essential for maximal steroidogenic activity (11), consistent with earlier studies demonstrating that StAR is a phosphoprotein (13,26). The recombinant proteins we employed were not phosphorylated.…”

Section: Discussionsupporting

confidence: 62%

“…Cyclic AMP influences StAR gene expression, like many proteins important for steroidogenesis (8 -10), and enhances StAR activity by triggering posttranslational modifications (11)(12)(13).…”

Section: Steroidogenic Acute Regulatory Protein (Star)mentioning

confidence: 99%

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The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Arakane

Kallen

Watari

et al. 1998

Journal of Biological Chemistry

178

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Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450 scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH 2 -terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidinetagged N-62 StAR (His-tag StAR) constructs lacking the NH 2 -terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose-and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

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Section: His-tag Star Proteinssupporting

confidence: 83%

Section: Discussionsupporting

confidence: 62%

See 1 more Smart Citation

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR)

Arakane

Kallen

Watari

et al. 1998

Journal of Biological Chemistry

178

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“…StAR is a phosphoprotein (4,5), and the phosphorylation state of StAR is directly correlated with steroidogenesis (16,17). We have previously shown that the ability of StAR to be phosphorylated at serine residue 195, which is in the context of a consensus protein kinase A phosphorylation motif that is conserved in all species of StAR identified to date, is associated with increased steroidogenic activity (18).…”

Section: Star Is a Sterol Transfer Protein 26287 Discussionmentioning

confidence: 99%

Steroidogenic Acute Regulatory Protein (StAR) Is A Sterol Transfer Protein

Kallen¹,

Billheimer²,

Summers³

et al. 1998

Journal of Biological Chemistry

194

122

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Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis by enhancing the delivery of substrate cholesterol from the outer mitochondrial membrane to the cholesterol side chain cleavage enzyme system on the inner membrane. A recombinant StAR protein lacking the first N-terminal 62 amino acid residues that includes the mitochondrial targeting sequence was shown to stimulate the transfer of cholesterol and ␤-sitosterol from liposomes to heat-treated mitochondria in a dose-, time-, and temperature-dependent manner. A recombinant mutant StAR protein that cannot stimulate steroidogenesis by isolated mitochondria did not promote sterol transfer. Unlike the more promiscuous lipid transfer protein, sterol carrier protein 2 (SCP 2 ), StAR did not stimulate phosphatidylcholine transfer in our assay system. The recombinant StAR protein increased cholesterol transfer to heattreated microsomes as well as to heat-and trypsintreated mitochondria. These observations demonstrate that StAR has sterol transfer activity, which may reflect an ability to enhance desorption of cholesterol from sterol-rich donor membranes. We suggest that the ability of StAR to promote sterol transfer explains its steroidogenic activity.

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“…It allows for the transfer of cholesterol from cytoplasmic pools via the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM) [3][4][5]. Under the influence of trophic hormones [6,7], StAR is acutely synthesized de novo in the cytoplasm as a 37-kDa protein. Due to its N-terminal import sequence [8,9], StAR is translocated into the mitochondria to be proteolytically cleaved to yield a 30-kDa protein [10].…”

Section: Introductionmentioning

confidence: 99%