http://www.unc.edu/courses/2009spring/envr/740/001 slide 1Begin 03/05/09Start lecture by reviewing the 2 overheads summarizing the genes involved in operation of extrinsic checkpoints in yeast.Row 1 lists the event triggering the extrinsic checkpoint and the response elicited, correlated with the point in the cell cycle where the event occurs (top panel). The second row identifies the genes involved. The next overhead will elaborate further on RAD17, RAD24 and MEC3 which detect damage in the G1, S and G2 phases. The third row indicates the action of the checkpoint. At the bottom of this overhead is a list of “effector” proteins, which are involved in initiating signaling pathways following the detection of damage. The genes highlighted in red have human analogues that were identified through studies of genetic diseases. The next overhead summarizes the cascade initiated in G1, S and G2 in yeast involving the RAD and MEC3 proteins: [OH]RAD17, RAD24 and MEC3 are presumed to bind to damage and activate transducer proteins MEC1 and TEL1, which are kinases whose targets are effector proteins RAD53 and Chk1 (Chk1 not on previous overhead) that in turn phosphorylate and activate check point proteins. Based on the description of the Ras pathway, the terms “transducer” and “effector” could be used interchangeably here. In humans, ATM is the homologue of TEL1, ATR is a homologue of MEC1, and Chk2 is a homologue of RAD53.The next overhead shows how DNA damage causes arrest in the G2 phase in higher organisms including humans, preventing progression to M phase until DNA damage is repaired.[OH]ATM which may serve both to detect damage and as signal transducer, activates kinase Chk2. 1This would correspond to the end point of the yeast cascade on previous overhead. This cascade goes further in describing the check point activation: Chk2 phosphorylates a protein called Cdc25 on Ser216. Cdc25 is responsible for the mediating dephosphorylation of Cdc2 (p34), whichyou remember from last lecture is necessary to allow transition into M phase. In the phosphorylated state, Cdc25 binds to a protein 14-3-3, represented in the overhead as the small sphere, which blocks phosphorylase activity, thus preventing M, phase from beginning.TUMOR SUPPRESSORSThe description of cell cycle checkpoints sets the stage for a discussion of tumor suppression. The loss of function of tumor suppressors represents a major pathway leading to the transformation of cells in culture or formation of tumors in vivo, and is the focus of our discussion. The protein functioning as a tumor suppressor at the earliest point in the cell cycle is RB, which is a substrate for cdk-cyclin D complexes and functions during the period of the G1 phase that precedes restriction (commitment to replication). The cartoon in the next slide shows that[OH1]during the initial period of G1, RB is bound to transcription factor E2F. Tying up E2F prevents transcription of genes whose products are essential for S phase, thereby blocking entry into S phase. The non-phosphorylated form of RB complexes with dimers of cdk4,6-cyclin D1,2,3 and also cdk2-cyclin E. Near the restriction point, cdk4,6-cyclin D complexes phosphorylate RB, which causes it to release E2F, permitting S phase to proceed. (There is some support for cyclin D1 as being the cyclin most important in deactivating RB).RB is a target for checkpoint pathways that inhibit growth. Several of the pathways act through inhibitory proteins called CKIs, which are the proteins in green in the cartoon. The cki proteins are classified in two families: the INK4 family specifically targets cdk4,6 and is comprised of four members: p16INK4A (which is shown in the cartoon), p15INK4B, p18INK4C and p19INK4D. (The proteins are named for size in lower case, and superscripted with trivial names, consult the text to2see this convention.) The pathway involving INK4 proteins is illustrated in the overhead for p16:[Genes VIII, Fig. 29.29] p16 binds next to an ATP binding site of cdk4 or 6, with the result that the catalytic activity is directly blocked and also an allosteric effect prevents cyclin from binding to the cdk catalytic unit, illustrated in the next overhead. Therefore, p16 prevents both assembly of cdk4,6-cyclin D dimer and its activity:[Genes VIII, Fig. 29.30]The Kip family indicated on the next overhead, inhibits all G1 and S phase cdk enzymes and consists of three members: p21Cip/WAF1, p27Kip1, shown in overhead of the RB cycle and p57Kip2. p21 is a universal cdk inhibitor, binding to all complexes of cdk2,4,6 (blocking activation by kinase CAK) and is therefore probably capable of blocking progression through all stages of G1/S. p21 is usually associated with cdks in a 1:1 stiochiometry along with PCNA (eukaryotic analogue of β-clamp for pol ) and by implication suggests involvement of p21 with regulation of transcription. In the 1:1 stiochiometry, p21 binding with cdk-cyclin dimers is not inhibitory. This effect is exhibited only when p21 is present in excess. p27 has a region of homology to p21,and appears to function in much the same manner, as the cartoon suggests:Because RB along with the CKIs are needed to suppress unrestrained growth, that they are all classified as tumor suppressors.APOPTOSISProgrammed cell death is a normal component of eukaryotic development. This process is called apoptosis, and it is critical to maintaining the proper level of cell population. Apoptosis can be triggered by various environmental stresses and damage to the cell and the inability of damaged cells to apoptose can result in development of tumors. Like the other processes we have 3described, apoptosis is a response to signals and involves initiation of cascades of events, which include the activation of proteins. The next slide illustrates some of the triggering events:[Genes VIII, Fig. 29.47]From the initial description of p53 as a tumor suppressor and the fact that p53 is also involved in multiple apoptotic pathways, this overhead indicates that p53 is a multifunctional protein, and that the pathways and mechanisms associated with p53 are complex. The next overhead is a simple-minded reminder that p53 functions both in the operation of check points and in apoptosisand reflects the current thinking that its function is dependent at least in part on the point in the cell cycle where it operates.[OH[I will begin by following the general organization in a CRT review that is posted on the course