Identification of a BRCA1-associated kinase with potential biological relevance

Abstract

A biochemical approach was used to identify proteins which interact with human BRCA1. Through this work, a kinase activity which co-puri®es with BRCA1 has been identi®ed. This kinase activity, which phosphorylates BRCA1 in vitro, was originally identi®ed in Sf9 insect cells but is also present in cells of human origin including breast and ovarian carcinoma cell lines. The BRCA1 kinase activity in vitro is associated with a fragment of BRCA1 encompassing amino acids 329 ± 435. This peptide is also phosphorylated in various human cell lines. A computer-assisted sequence analysis revealed that this peptide was a potential substrate for phosphorylation by PKA, PKC, or CKII. However, phosphorylation by these kinases could not be demonstrated in vitro indicating the presence of another kinase activity. Phosphorylation in vitro requires a minimal domain of BRCA1 encompassing amino acids 379 ± 408. Notably, deletion of this minimal domain abolishes growth suppression by BRCA1 indicating that this domain, as well as phosphorylation within this domain, may be important for BRCA1 function.

BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

“…However, our previous studies revealed that BRCA1 is predominantly phosphorylated on serine and threonine residues, at least in the cell lines analyzed (57). Furthermore, a kinase activity that associates with and phosphorylates a BRCA1 fragment containing aa 329 to 435 in vitro was identified (9).…”

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

“…The following mutagenic primers were used: 5 -C ACA CGA GGG CAG CCT GTC CTC GAG CCA CCA GAT CAG CTG GTC-3 (for αT638E); 5 -C CAG TCT GAT TTT GAA GGG TTC GAA TAT GTC AAC CCC CAG TTT GTG C-3 (for αT657E); 5 -C GAT GAG GTT CTT GTC ACT GAA CTC GAG TTG GGG TTT CTC ATT CAG G-3 (for δS643E); 5 -G CTC ATA TTT GGG GTT CAC GAA TTC GAA GCC CTT GAA GGC TGT CTG G-3 (for δS662E). GFP-tagged deletion mutants of PKCα and PKCδ lacking the C1A or C1B domain were constructed using QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) [26] with PKCα-GFP or PKCδ-GFP as a template. The primers used for the deletion were as follows: 5 -G AAG AAC GTG CAT GAG GTG AAA GAC CCG GGT GCG GAT AAG GGA CCT GAC-3 and 5 -GTC AGG TCC CTT ATC CGC ACC CGG GTC TTT CAC CTC ATG CAC GTT CTT C-3 (for αC1A); 5 -GAC ACT GAT GAC CCC AGA AGC AAG GGA ATG GAT CAC ACA GAG AAG AGG-3 and 5 -CCT CTT CTC TGT GTG ATC CAT TCC CTT GCT TCT GGG GTC ATC AGT GTC-3 (for αC1B); 5 -GCC AAG ATT CAC TAC ATC AAG AAC ACT GGC ACT GCT ACC AAT AGC CGG G-3 and 5 -C CCG GCT ATT GGT AGC AGT GCC AGT GTT CTT GAT GTA GTG AAT CTT GGC-3 (for δC1A).…”

Gene therapy for carcinoma of the breast: Therapeutic genetic correction strategies

“…Although the molecular function of BRCA1 is controversial and may include DNA repair or transcriptional functions [13], overexpression of BRCA1 into sporadic breast or ovarian cancer cells, which usually show low BRCA1 expression [14], results in growth inhibition and tumor suppression [15,16,17,18,19,20,21,22,23]. Growth inhibition by BRCA1 has itself been controversial, leading some to question the rationale for BRCA1 gene therapy [24], but subsequent studies have shown growth inhibition or tumor suppression by BRCA1 in vitro [16,17,19,20,21,22,23] and in vivo [15,17,21,25].…”

“…Growth inhibition by BRCA1 has itself been controversial, leading some to question the rationale for BRCA1 gene therapy [24], but subsequent studies have shown growth inhibition or tumor suppression by BRCA1 in vitro [16,17,19,20,21,22,23] and in vivo [15,17,21,25]. The mechanism of growth inhibition by BRCA1 is unknown and may involve interactions with WAF1/CIP1 [16], p53 [17], Rb [23] or induction of apoptosis [18,19].…”