Question

Name 2ors L! You are inte finding protein are interested in understanding how Top5 expression is regulated. Top5 is a microtubule protein. You study a cell line where Top5 expression is controlled by a signal, Ligl. When hinds to and activates the membrane bound recentor it activates the Ras protein, which activates atein kinase called PKF, which then phosphorylates Grp4 on T224. Grp4 is a gene regulatory Wotein that can only enter the nucleus when phosphorylated: Thos is not expressed when Grp4 is not the nucleus. Grp4 binds to the regulatory sequences of the The Rene and activates transcription. This pathway is diagrammed in the Figure here. Top5 is not expressed when Grp4 is not in the nucleus. plasma membrane receptor 1) For each of the following members of this pathway, indicate the location of the ribosomes that translate each of these proteins. (2 points cach) activated a) the Ligl receptor Grpt b) Tbp5 This gone NUCLEUS c) Grp4 The's protein in) State whether you would see Top5 mRNA being produced or not produced in the conditions below. if cells were treated with Ligl. (2 points each) a) Addition of a drug that inhibits the ability of Ras to hydrolyze GTP. b) If PKF was constitutively active. c) If normal Grp4 was replaced with Grp4 that had T224 changed to alanine. d) Replacement of normal Ras with a constitutively active Ras. ill) If you were to take the cells that have constitutively active Ras (Q2, partiid) and add a drug that prevents the receptor from binding to Ligl, how do you predict this would affect Top5 expression when Ligl is present? Explain. (4 points) ame

Answer 1

According to the given data, few points are summarized.

Tbp5 protein is a microtubule binding protein. Its expression is controlled by Lig 1. It is a signal molecule.

Lig1 binds to its receptor on the plasma membrane. Soon it binds, activates it and this in turn activates the Ras protein in the cytoplasm.

The activated Ras protein activates a protein kinase called PKF. Activated PKF phosphorylates the Grp4 protein. It is a gene regulatory protein.

Phosphorylated Grp4 enters into nucleus from cytoplasm. In the nucleus it binds to regulatory sequences of Tbp5 gene and allows transcription of that gene.

Hence, mRNA is produced inside the nucleus. The formed mRNA is transported to cytoplasm and translated in to a protein in cytoplasm.

ii) in first condition (a) a drug is added that inhibits the ability of Ras to hydrolyze GTP. In this condition, if GTP is not hydrolyzed, the phosphorylation of Grp4 cannot take place. So, Grp4 cannot enter into the nucleus, if it is not phosphorylated. Therefore, no binding of Grp4 to regulatory sequences of tbp5 gene. Thus mRNA is not produced.

(b) if PFK was active, it phosphorylates Grp4 protein. Thus, transcription is activated producing Tbp5 mRNA.

(c) replacing normal Grp4 with Grp4 having T224 changed to alanine, then phosphorylation will not occur as T224 is not available. So, mRNA is not produced as Grp4 cannot enter the nucleus without phosphorylation.

(d) normal Ras replaced by constitutive active Ras, then phosphorylation of Grp4 occurs continuously. So, Tbp5 mRNA is produced  as phosphorylated Grp4 enters the nucleus and binds with gene regulatory sequences of Tbp5 gene.

iii) Lig1 is a signal molecule that binds to receptor present on the membrane and activates it. by this time, Ras protein will be in an inactivated form. Binding of signal molecule to the receptor activates the Ras protein. In the above given condition, Ras protein is constitutive active. The cells are provided with a drug that prevents the binding of Lig1 to receptor. So, at this condition the expression of Tbp5 gene is not affected as Ras is active in cytoplasm irrespective of binding of signal molecule to receptor. So, Tbp5 protein will be produced.