Dr Mohammad S Alanazi, MSc, PhD Molecular Biology KSU Cell Cycle Control, Defects and Apoptosis 1 st Lecture

The Cell Cycle Is an Ordered Series of Events Leading to Replication of Cells 2 Cell-Cycle Control in Mammalian Cells

Dr Gihan Gawish 3 Regulated Protein Phosphorylation and Degradation Control Passage through the Cell Cycle

 Amphibian and invertebrate eggs and early embryos from synchronously fertilized eggs provide sources of extracts for biochemical studies of cell-cycle events.  The isolation of yeast cell-division cycle (cdc) mutants led to the identification of genes that regulate the cell cyclegenescell cycle Diverse Experimental Systems Have Been Used to Identify and Isolate Cell-Cycle Control Proteins 4

Dr Gihan Gawish 5 Isolation of wild-type cell-division cycle (CDC) genes from S. cerevisiae cells carrying temperature-sensitive mutations in these genes

6  In multicellular organisms, cell replication is controlled by a complex network of signaling pathways that integrate signals from the extracellular environment with intracellular cues about cell size and developmental program.  Polypeptide growth factors called mitogens stimulate cultured mammalian cells to cycle. Once cycling cells pass the restriction point, they can enter the S phase and complete S, G 2, and mitosis in the absence of growth factors. Mammalian Restriction Point is Analogous to start in Yeast Cells

7 Multiple Cdks and Cyclins Regulate Passage of Mammalian Cells through the Cell Cycle Experimental demonstration that cyclin D is required for passage through the restriction point in the mammalian cell cycle

8 Multiple Cdks and Cyclins Regulate Passage of Mammalian Cells through the Cell Cycle Activity of mammalian Cdkcyclin complexes through the course of the cell cycle in G 0 cells induced to divide by treatment with growth factors The width of the colored bands is approximately proportional to the protein kinase activity of the indicated complexes. Cyclin D refers to all three D-type cyclins.

Dr Gihan Gawish 9

10 The Cell-Cycle Control System

11 GENERAL NAMEFUNCTIONS AND COMMENTS Protein kinases and protein phosphatases that modify Cdks Cdk-activating kinase (CAK) phosphorylates an activating site in Cdks Wee1 kinasephosphorylates inhibitory sites in Cdks; primarily involved in controlling entry into mitosis Cdc25 phosphatase removes inhibitory phosphates from Cdks; three family members (Cdc25A, B, C) in mammals; Cdc25C is the activator of Cdk1 at the onset of mitosis Cdk inhibitory proteins (CKIs) Sic1 (budding yeast)suppresses Cdk activity in G 1 ; phosphorylation by Cdk1 triggers its destruction p27 (mammals) suppresses G 1 /S-Cdk and S-Cdk activities in G 1 ; helps cells to withdraw from cell cycle when they terminally differentiate; phosphorylation by Cdk2 triggers its ubiquitylation by SCF p21 (mammals)suppresses G 1 /S-Cdk and S-Cdk activities following DNA damage in G 1 ; transcriptionally activated by p53 p16 (mammals)suppresses G 1 -Cdk activity in G 1 ; frequently inactivated in cancer Ubiquitin ligases and their activators SCF catalyzes ubiquitylation of regulatory proteins involved in G 1 control, including CKIs (Sic1 in budding yeast, p27 in mammals); phosphorylation of target protein usually required for this activity APC catalyzes ubiquitylation of regulatory proteins involved primarily in exit from mitosis, including Securin and M-cyclins; regulated by association with activating subunits Cdc20 APC-activating subunit in all cells; triggers initial activation of APC at metaphase-to- anaphase transition; stimulated by M- Cdk activity Hct1maintains APC activity after anaphase and throughout G 1 ; inhibited by Cdk activity Gene regulatory proteins E2F promotes transcription of genes required for G 1 /S progression, including genes encoding G 1 /S cyclins, S-cyclins, and proteins required for DNA synthesis; stimulated when G 1 -Cdk phosphorylates Rb in response to extracellular mitogens p53 promotes transcription of genes that induce cell cycle arrest (especially p21) or apoptosis in response to DNA damage or other cell stress; regulated by association with Mdm2, which promotes p53 degradation The Major Cell-cycle Regulatory Proteins

12  In multicellular organisms, cells that are no longer needed or are a threat to the organism are destroyed by a tightly regulated cell suicide process known as programmed cell death, or apoptosis.  Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus.  Caspases exist in all cells as inactive precursors, or procaspases, which are usually activated by cleavage by other caspases, producing a proteolytic caspase cascade.  The activation process is initiated by either extracellular or intracellular death signals, which cause intracellular adaptor molecules to aggregate and activate procaspases. Caspase activation is regulated by members of the Bcl-2 and IAP protein families.  In multicellular organisms, cells that are no longer needed or are a threat to the organism are destroyed by a tightly regulated cell suicide process known as programmed cell death, or apoptosis.  Apoptosis is mediated by proteolytic enzymes called caspases, which trigger cell death by cleaving specific proteins in the cytoplasm and nucleus.  Caspases exist in all cells as inactive precursors, or procaspases, which are usually activated by cleavage by other caspases, producing a proteolytic caspase cascade.  The activation process is initiated by either extracellular or intracellular death signals, which cause intracellular adaptor molecules to aggregate and activate procaspases. Caspase activation is regulated by members of the Bcl-2 and IAP protein families. Programmed Cell Death (Apoptosis)

Dr Gihan Gawish 13 (A) Each suicide protease is made as an inactive proenzyme (procaspase), which is usually activated by proteolytic cleavage by another member of the caspase family (B) Each activated caspase molecule can cleave many procaspase molecules, thereby activating them, and these can then activate even more procaspase molecules. (A) Each suicide protease is made as an inactive proenzyme (procaspase), which is usually activated by proteolytic cleavage by another member of the caspase family (B) Each activated caspase molecule can cleave many procaspase molecules, thereby activating them, and these can then activate even more procaspase molecules. The caspase cascade involved in apoptosis

Dr Gihan Gawish 14 Procaspases Are Activated by Binding to Adaptor Proteins

15 The factors that promote organ or organism growth can be operationally divided into three major classes:  Mitogens, which stimulate cell division, primarily by relieving intracellular negative controls that otherwise block progress through the cell cycle.  Growth factors, which stimulate cell growth (an increase in cell mass) by promoting the synthesis of proteins and other macromolecules and by inhibiting their degradation.  Survival factors, which promote cell survival by suppressing apoptosis. Extracellular Control of Cell Division, Cell Growth, and Apoptosis The extracellular signal molecules that regulate cell size and cell number are generally either soluble secreted proteins, proteins bound to the surface of cells, or components of the extracellular matrix.