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Slavova-Azmanova N.S.,University of Western Australia | Kucera N.,University of Western Australia | Louw A.,University of Western Australia | Louw A.,Royal Perth Hospital Medical Research Foundation | And 9 more authors.
Biochemical Journal | Year: 2014

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn-/- erythroblasts had attenuated ex vivo expansion, and late stage Lyn-/-erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn-/-erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKa/ß (glycogen synthase kinase a/ß) phosphorylation and diminished p27Kip1 induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro-and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling. © The Authors Journal compilation © 2014 Biochemical Society.

Slavova-Azmanova N.S.,University of Western Australia | Kucera N.,University of Western Australia | Satiaputra J.,University of Western Australia | Stone L.,University of Western Australia | And 8 more authors.
Blood | Year: 2013

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lynup/up) on erythropoiesis and Epo receptor signaling. Adult Lynup/up mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn+/up mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn+/up erythroid cell lines and primary CD71+ Lynup/up erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated- binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn+/up cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects. © 2013 by The American Society of Hematology.

Whiting R.J.,University of Western Australia | Payne C.J.,University of Western Australia | Payne C.J.,Royal Perth Hospital Medical Research Foundation | Satiaputra J.,University of Western Australia | And 7 more authors.
Biochemical Journal | Year: 2012

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation ofLyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn. © The Authors Journal compilation © 2012 Biochemical Society.

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