Huot, Thomas Jocelyn Georges; (2003) Interplay of Id and INK4 proteins in cellular senescence. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This study evaluates the functional interplay of two diverse families of proteins, both of which have critical roles in regulating cell proliferation and differentiation. The Id family, of which there are four known members (Idl-Id4), are helix-loop-helix (HLH) proteins that act as dominant negative inhibitors of basic HLH (bHLH) transcription factors by forming heterodimers that are unable to bind DNA. However, it has recently been recognised that they also influence the activity of the Ets family of transcription factors. Moreover, Id2 and Id3 have been shown to undergo phosphorylation in late G1/early S phase of the cell cycle on a serine residue (Ser5) within a consensus target site for cyclin-dependent kinases (CDKs). The initial phase of this work extended the observation to Id4 and examined the functional consequences of phosphorylation. In a model electrophoretic mobility shift assay, phosphorylation on Ser5 either negates or alters the specificity of competitive interactions between Id proteins and bHLH homo- or heterodimers. The second family of proteins are the INK4 cyclin-dependent kinase inhibitors which act on CDK4 and CDK6, the kinases that initiate phosphorylation of the retinoblastoma protein (pRb) during the transition from G1 to S phase. The prototype of this family, pl6INK4a, is implicated in replicative senescence either in response to telomere exhaustion or as a consequence of oncogenic challenge. In a collaborative study, it was established that the Ras/Raf/MEK signalling pathway activates expression via the Ets2 transcription factor and that this effect is counteracted by Id proteins. Although Id1 appears to be the major player in vivo, Id2 is equally effective in vitro. A model is proposed in which the phosphorylation of Id2 by CDK2 overrides its ability to oppose Ets binding to p16INK4a promoter. A role in senescence may explain why p16INK4a is an important tumour suppressor. As well as being a frequent target of mutations in sporadic cancers, germline mutations of p16INK4a are associated with predisposition to malignant melanoma. As part of an ongoing survey of germline alterations, a patient was identified that had inherited separate missense mutations on each allele of CDKN2A, the locus that encodes p16INK4a Both mutations affect the amino acid sequence of p16INK4a but only one changes the sequence of p14ARF , the alternative product specified by CDKN2A. Biochemical and functional analyses indicated that the two p16INKA variants are either totally or partially defective whereas the p14ARF variant remains fully functional. These data reinforce the view that p16INK4A is the critical tumour suppressor in malignant melanoma and establish that HDFs from this individual are specifically defective for p16INK4a. These HDFs were used to extend the analyses of Ets-mediated induction of p16iNK4a as well cellular transformation by oncogenic Ras.

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