br Expression of Lyn kinase is regulated
3.6. Expression of Lyn kinase is regulated by SRC-2
Although SRC-2 and SRC-3 depletions in MCF-7 Z-VAD-FMK induced si-milar changes in the cell phenotype in 3D cultures (Fig. 4), the majority of genes diﬀerentially expressed by SRC-2 and SRC-3 knock-down were unique to each of the two proteins (Fig. 2). In a search for additional SRC-2-specific inducers of diﬀerentiation, we ranked SRC-2 unique genes based on fold of diﬀerential expression (Table 3). Amongst the most diﬀerentially expressed genes we identified up-regulations of HEY2, which was shown by RT-qPCR to be upregulated by SRC-3 as well (Fig. 5), in addition to the well-known pluripotency gene SOX2, and the breast cancer EMT promoter Lyn kinase (Table 3, highlighted). We therefore further analyzed the expression of Lyn kinase in shMCF-7 cells by RT-qPCR and immunoblotting (Fig. 7). In line with our mi-croarray results, we observed an increased expression of total Lyn mRNA (Fig. 7a) and protein (Fig. 7b, up) in cells with SRC-2 knock-down, whereas no change was observed in the shSRC-3 cells. Further-more, we detected changes in the phosphorylation pattern of two Lyn isoforms (Lyn56 and Lyn53) on the residue Tyr507 (Fig. 7b). Lyn phosphorylated on Tyr507 has a minimum basal activity and is avail-able to be fully activated by a phosphorylation of its Tyr397 residue . As shown in Fig. 7b, shKTR and shSRC-3 cells feature higher levels of the phosphorylated longer 56-kD Lyn isoform, Lyn56, than they do of
O. Bozickovic et al.
Table 3 Most diﬀerentially expressed SRC-2 unique genes.
Gene Symbol Protein Name Fold
FXYD4 FXYD domain containing ion transport regulator 4 6,11 SPRR3 small proline-rich protein 3 4,52 OXTR oxytocin receptor 2,47 RPRM reprimo, TP53 dependent G2 arrest mediator 2,36
CA2 carbonic anhydrase II 2,29 KLK8 kallikrein related-peptidase 8 2,17 PODXL podocalyxin-like 2,09 HEY2 hairy/enhancer-of-split related with YRPW motif 2 2,07 UPK2 uroplakin 2 2,06 SOX2 SRY (sex determining region Y)-box 2 1,99 C19orf33 chromosome 19 open reading frame 33 1,94 CDH26 tv a cadherin 26, transcript variant a 1,90 CSRP2 cysteine and glycine-rich protein 2 1,86 PKIA protein kinase (cAMP-dependent, catalytic) 1,83
IGFBP2 insulin-like growth factor binding protein 2, 36kDa 1,83 PAPSS2 3'-phosphoadenosine 5'-phosphosulfate synthase 2 1,83 LYN LYN proto-oncogene, Src family tyrosine kinase 1,82 FAM46 A family with sequence similarity 46, member A 180 FAM217B family with sequence similarity 217, member B 1,79 CXCR4 chemokine (C-X-C motif) receptor 4 1,76 Down-regulated
G6PD tv1 glucose-6-phosphate dehydrogenase, transcript −2,83
ITM2C tv2 integral membrane protein 2C, trancript variant 2 −2,28 SPG21 tv1 spastic paraplegia 21, transcript variant 1 −2,17 MGP matrix Gla protein −2,01 GNS glucosamine (N-acetyl)-6-sulfatase −1,96 YIF1B tv2 Yip1 interacting factor homolog B, transcript variant −1,96
GSTM1 glutathione S-transferase mu 1 −1,95 SUN2 Sad1 and UNC84 domain containing 2 −1,94 SLC7A5 solute carrier family 7 (amino acid transporter light −1,91
FAIM3 Fas apoptotic inhibitory molecule 3 −1,88 SMARCD3 SWI/SNF related, matrix associated, actin −1,86
dependent regulator of chromatin, subfamily d,
IFI27 interferon, alpha-inducible protein 27 −1,85 HSPB8 heat shock 22 kDa protein 8 −1,78 SBK1 SH3 domain binding kinase 1 −1,76 NAV2 neuron navigator 2 −1,74 IRF9 interferon regulatory factor 9 −1,73 YIF1B tv4 Yip1 interacting factor homolog B, transcript variant