<i>PRKAR1A</i> Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

Robinson‐White, Audrey; Leitner, Wolfgang W.; Aleem, Eiman; Kaldis, Philipp; Bossis, Ioannis; Stratakis, Constantine A.

doi:10.1158/0008-5472.can-06-2200

Cited by 37 publications

(35 citation statements)

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“…© 2008 American Association for Cancer clincancerres.aacrjournals.org Downloaded from premature stop codon generation and subsequent nonsensemediated mRNA decay. Our experiments with cells bearing these mutations showed elevation of the total cAMP-stimulated kinase activity, consistent with the phenotype of PRKAR1A haploinsufficiency (4,13,16). It is unlikely that this deletion represents a rare copy number variation because it was not present in the limited number of control DNA samples that we tested but also because of its correlation with the Carney complex phenotype.…”

Section: Discussionsupporting

confidence: 80%

“…The relatively milder in vivo effect in lymphocytes is most likely due to the predominantly expressed (under a condition of heterozygosity) wt allele (see Fig. 2C); this is not unexpected because lymphocytes are not affected by Carney complex and the message for the wt PRKAR1A allele remains strongly expressed (13).…”

Section: Resultsmentioning

confidence: 83%

“…Blood samples were collected from patients, as previously described (5). Lymphocytes were separated from whole blood and transformed by EBV, and the cell cultures were maintained in RPMI 1640 with 1% L-glutamine, 10% fetal bovine serum, and 1% antibiotic/antimycotic agents (13). DNA, RNA, and proteins were extracted from whole blood and cell cultures using standard procedures.…”

Section: Methodsmentioning

confidence: 99%

“…PKA enzymatic activity was measured by a method described previously (13). The assays in a total volume 50 AL were carried out for 15 min at 37jC in the reaction mixture containing 1 mol/L Tris-HCl (pH 7.5), 1 mol/L DTT, 1 mol/L MgCl 2 , 60 Amol/L Kemptide (a phosphate acceptor peptide; Leu-Arg-Arg-Ala-Ser-Leu-Gly), 20 Amol/L [g-32 P]ATP (25 Ci/mmol), with or without 5 Amol/L cAMP and 100 Amol/L PKA inhibitor (PKI), and 10 AL of the cell extracts.…”

Section: Methodsmentioning

confidence: 99%

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Large Deletions of the PRKAR1A Gene in Carney Complex

Horvath

Bossis

Giatzakis

et al. 2008

Clinical Cancer Research

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Purpose: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in f70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A^mutation-negative individuals with Carney complex. Experimental Design: To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2. Results: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma. Conclusions: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.Carney complex (MIM 160980) is an autosomal dominant multiple endocrine neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, and different types of endocrine tumors, including multiple hyperfunctioning adrenal nodules, growth hormone -secreting pituitary tumors, and gonadal and thyroid neoplasias; rarely, malignant psammomatous melanotic schwannomas are present (1 -3).Inactivating mutations in the PRKAR1A gene coding for the type 1A regulatory subunit of protein kinase A [PKA; cyclic AMP (cAMP)-dependent protein kinase] have been shown to cause the disease (4, 5). To date, 60 different PRKAR1A pathogenic mutations have been described; these are mostly single base pair (bp) substitutions and some exonic insertions and deletions that are detectable by PCR-based approaches (5 -9).

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Section: Discussionsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Large Deletions of the PRKAR1A Gene in Carney Complex

Horvath

Bossis

Giatzakis

et al. 2008

Clinical Cancer Research

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“…31 Notably, activation of the H-Ras signalling pathway has been implicated in excessive cell proliferation caused by Prkar1a haploinsufficiency in human patients. 7,32 We used ROSACreER T2 ;Prkar1a fl/ þ for this experiment because ROSACreER T2 ;Prkar1a fl/fl injected with tamoxifen died within a week due to cachexia. ROSA-CreER T2 , ROSACreER T2 ;Bim À / À , ROSA-CreER T2 ;Prkar1a fl/ þ and ROSACreER T2 ;Prkar1a fl/ þ ;Bim À / À mice (n ¼ 6 in each genotype) were injected with tamoxifen (40 mg/kg for three consecutive days) followed by a single topical application of DMBA.…”

Section: Resultsmentioning

confidence: 99%

Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice

et al. 2014

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Loss of function mutations in the Prkar1a gene are the cause of most cases of Carney complex disorder. Defects in Prkar1a are thought to cause hyper-activation of PKA signalling, which drives neoplastic transformation, and Prkar1a is therefore considered to be a tumour suppressor. Here we show that loss of Prkar1a in genetically modified mice caused transcriptional activation of several proapoptotic Bcl-2 family members and thereby caused cell death. Interestingly, combined loss of Bim and Prkar1a increased colony formation of fibroblasts in culture and promoted their growth as tumours in immune-deficient mice. Apart from inducing apoptosis, systemic deletion of Prkar1a caused cachexia with muscle loss, macrophage activation and increased lipolysis as well as serum triglyceride levels. Loss of single allele of Prkar1a did not enhance tumour development in a skin cancer model, but surprisingly, when combined with the loss of Bim, caused a significant delay in tumorigenesis and this was associated with upregulation of other BH3-only proteins, PUMA and NOXA. These results show that loss of Prkar1a can only promote tumorigenesis when Prkar1a-mediated apoptosis is somehow countered.

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Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update

et al. 2010

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PRKAR1A encodes the regulatory subunit type 1-alpha (RIa) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIa haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIa haploinsufficiency, but also by the expression of altered RIa protein, as proven by analysis of expressed mutations in the gene, consisting of aminoacid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed-these are frame-shifts in the 3 0 end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation-negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype. Hum Mutat 31:369-379,

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PRKAR1A Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

Cited by 37 publications

References 50 publications

Large Deletions of the PRKAR1A Gene in Carney Complex

Large Deletions of the PRKAR1A Gene in Carney Complex

Loss of Prkar1a leads to Bcl-2 family protein induction and cachexia in mice

Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update

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