Continuation of cell division regulation;

The signal transduction mechanism that leads to the conversion of a regulatory gene to a
viral oncogene
 A normal cell is infected by a retrovirus which;
 Inserts its own genome into the chromosome of the host cell, near the gene for a
regulatory protein (the proto-oncogene).
 The viral particles released from the infected cell sometimes “capture” a host gene, in this
case a proto-oncogene.
 During several cycles of infection, a mutation occurs in the viral proto-oncogene,
converting it to an oncogene.
 When the virus subsequently infects a cell, it introduces the oncogene into the cell’s
DNA.
 Transcription of the oncogene leads to the production of a defective regulatory protein
that continuously gives the signal for cell division.
 This overriding normal regulatory mechanisms.
 The host cells infected with oncogene-carrying viruses undergo unregulated cell division;
they form tumors.
 Proto-oncogenes can also undergo mutation to oncogenes without the intervention of a
retrovirus.

APOPTOSIS
The cascade of initial events of apoptosis:

 The regulatory mechanisms that trigger apoptosis involve some of the same proteins that
regulate the cell cycle.
 The signal for suicide often comes from outside, through a surface receptor.
 Tumor necrosis factor (TNF), produced by cells of the immune system, interacts with
cells through specific TNF receptors.
 These receptors have TNF-binding sites on the outer face of the plasma membrane and a
“death domain” of about 80 amino acid residues that passes the self-destruct signal

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 through the membrane to cytosolic proteins such as TRADD (TNF receptor-associated
death domain).
 Another receptor, Fas, has a similar death domain that allows it to interact with the
cytosolic protein FADD (Fas-associated death domain), which activates a cytosolic
protease called caspase 8.
 This enzyme belongs to a family of proteases that participate in apoptosis; all are
synthesized as inactive proenzymes, all have a critical Cys residue at the active site;
 And all hydrolyze their target proteins on the carboxyl-terminal side of specific Asp
residues (hence the name caspase).
 When caspase 8, an “initiator” caspase, is activated by an apoptotic signal carried through
FADD, it further self-activates by cleaving its own proenzyme form.
 Mitochondria are one target of active caspase 8.
 The protease causes the release of certain proteins contained between the inner and outer
mitochondrial membranes cytochrome c and several “effector” caspases.
 Cytochrome c binds to the proenzyme form of the effector enzyme caspase 9 and
stimulates its proteolytic activation.
 The activated caspase 9 in turn catalyzes wholesale destruction of cellular proteins—a
major cause of apoptotic cell death.
 One specific target of caspase action is a caspase-activated deoxyribonuclease. In
apoptosis, the monomeric products of protein and DNA degradation (amino acids and
nucleotides) are released in a controlled process that allows them to be taken up and
reused by neighboring cells.
 Apoptosis thus allows the organism to eliminate a cell without wasting its components.

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 Initial events of apoptosis

AUTOPHAGY

The autophagy pathway in eukaryotes:

 Autophagy is a stress-induced catabolic process involving the lysosome (or, in yeast, the
analogous vacuole), which is conserved in all eukaryotes.
 According to the different pathways by which cargo is delivered to the lysosome or
vacuole, autophagy is divided into three main types: chaperone-mediated autophagy
(CMA), microautophagy and macroautophagy.
 CMA is a process that has been characterized in higher eukaryotes but not in yeast. In
CMA, a chaperone protein binds first to its cytosolic target substrate and then to a
receptor on the lysosomal membrane where the unfolding of the protein occurs.

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 The unfolded cytosolic target protein is subsequently translocated directly into the
lysosome for its degradation.
 Microautophagy translocates cytoplasmic materials into the lysosome or vacuole for
degradation by either direct invagination, protrusion, or septation of the lysosomal or
vacuolar membrane.
 Macroautophagy is characterized by the formation of a cytosolic double-membrane
vesicle, the autophagosome.
 During macroautophagy, cytoplasmic proteins, organelles or other materials are
surrounded by phagophores, which expand and close to form autophagosomes.
 These autophagosomes fuse with lysosomes (or vacuoles) to form autolysosomes, in
which the cytoplasmic cargos are degraded by resident hydrolases.
 The resulting degradation products are then transported back into the cytosol through the
activity of membrane permeases for reuse.
 The process and mechanism of microautophagy in mammalian cells are still not clear.
Among the three main forms of autophagy, macroautophagy is the most widely studied
and best characterized process.

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 The autophagy pathway

The importance of autophagy in cell survival and cell death:

 The primary role of autophagy is to protect cells under stress conditions, such
as starvation.
 During periods of starvation, autophagy degrades cytoplasmic materials to produce
amino acids and fatty acids that can be used to synthesize new proteins or are oxidized by
mitochondria to produce ATP for cell survival.
 However, when autophagy is excessively induced, it can result in autophagic cell death,
so-called type II programmed cell death (PCD), which is distinct from type I PCD
(apoptosis) and from necrosis.
 In addition to stress management, autophagy is involved in normal development,
senescence, lifespan extension, immunity and defense against microbial invasion.

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  Autophagy also has a role in many human pathophysiologies, such as cancer,
myopathies, neurodegeneration, heart and liver diseases, and gastrointestinal disorders.

The crosstalk between autophagy and apoptosis in mammalian cells;

 Autophagic degradation of active caspase-8, a positive effector of apoptosis, is
responsible for the inhibition of apoptotic cell death in mammalian cells.
 The pro-cell death effect of autophagy could be related to the activation of apoptosis,
which would imply that autophagy is an upstream event of apoptosis or, alternatively,
 autophagy could be independent of apoptosis, such as in situations, in which autophagy-
induced cell death does not exhibit any of the characteristic apoptotic features, such as
caspase activation and DNA fragmentation.
 Thus, it has been controversial as to how ‘autophagic cell death’ should be defined and,
for consistency, we here define it as ‘cell death that is triggered by autophagy’ (pro-cell
death effect).
 In contrast to the above-mentioned mechanism for the cytoprotective effect of autophagy
against stress, one possible mechanism for autophagic cell death could involve the
autophagic degradation of a negative effector of apoptosis.
 Factors that make it possible to differentiate the pro-cell survival and pro-cell death
effects of autophagy have been reported recently. For instance, Draper (Drpr), the D.
melanogaster ortholog of the C. elegans engulfment receptor CED-1 and Jun N-terminal-
kinase (JNK) are involved in autophagic cell death, but not in autophagy-induced cell
survival.
 Furthermore, death-associated protein kinase (DAPK) converts autophagy from a cell
survival mechanism to one of the initiation of cell death. It remains to be investigated
how exactly these factors (Drpr, JNK, DAPK, etc.) are able to direct autophagy from a
survival to a death pathway.
 Several Atg proteins have been Several Atg proteins have been implicated in apoptosis.
For example, caspase 3 cleaves the human Atg4 family member Atg4D to generate a
truncated product, DN63 Atg4D that, when overexpressed, induces autophagy-
independent apoptosis.

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  Similarly, under conditions that induce apoptosis, Atg5 is cleaved by calpains, generating
a truncated product 24K Atg5 that, when overexpressed by itself, induces apoptosis, but
not autophagy.
 Caspases also cleave beclin 1 at Asp149 during apoptosis resulting in the inhibition of
autophagy.

Figure: Crosstalk between autophagy and apoptosis in mammalian cells. (A) In a cell, the
same stress induces both autophagy and apoptosis as independent processes. A stress can also
induce autophagy, which then inhibits apoptosis or, alternatively, a stress induces autophagy,
which can be the trigger of apoptosis (bottom). (B) Both autophagy and apoptosis are negatively
regulated by Bcl-2, Bcl-xL and Flip. (C) Autophagy proteins Atg5,beclin 1 and Atg4D function
in autophagy in their unmodified form, but also have a role in apoptosis after cleavage by either
calpains, which target Atg5 and give rise to 24 K Atg5, or caspase 3, which cleaves beclin 1,
resulting in a C-terminal fragment of beclin 1 (Beclin 1-C), and Atg4D, truncating it at the
canonical caspase cleavage sequence (DEVD63K) to DN63 Atg4D.

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 MECHANISM OF ACTION OF HORMONES

Hormones can be classified as;

i. Steroids

 Steroids, whether from the adrenal, testes or ovaries, act through a common mechanism.
 They are all hydrophobic molecules and are thought, therefore, to be able to pass through
the lipid portion of the cell membrane without the need of a specific carrier or transport
protein.
 Once inside the cell they bind to specific receptors, and the steroid-receptor complex then
binds to specific acceptor sites on the nuclear matrix.
 Activation of the nuclear acceptor sites results in the initiation of transcription and the
production of new mRNA.
 The mRNA is translated in the cytoplasm to produce new protein.
 All steroids, therefore, act by stimulating the production of proteins.
 These new proteins then modify cell function. This is a relatively slow process; steroids
require several hours to alter cell function.

ii. Non-steroids
 The also belong to the class of peptide hormones. This includes amino acid derivatives,
prostaglandins, peptides, proteins, and glycoproteins.
 These molecules share a similar characteristic in that they cannot pass through the lipid
portion of the cell membrane.
 In order to be able to carry out their role as metabolic regulators, they must bind to
specific receptor molecules on the cell membrane and thereby transmit a message to the
interior of the cell.
 There are 2 types of receptor systems which these “peptide” hormones utilize. System
No. 2 is the classic second messenger system of which the cAMP system is the prototype.
 This system has 3 components: a receptor which is specific for a particular hormone, a
transducer molecule which recognizes an activated receptor (i.e., hormone-receptor
complex), and an effector molecule.
 When the hormone binds to the receptor on the cell surface, this hormone-receptor
complex is able to couple to and activate the transducer.
 The transducer, when activated, performs 2 tasks: it causes the hormone-receptor
complex to dissociate (thereby allowing the process to start again) and at the same time
couples to and activates the effector.
 This is a self- limiting process which is extremely short-lived and apparently results in
the transducer and the effector returning to their original state ready to be stimulated
again.

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  The effector faces the inside of the cell (or may be an integral protein which passes
through the entire membrane), and when activated, increases the concentration of the
second messenger.
 The effector can be an enzyme which, when activated, synthesizes the second messenger
or part of an ion channel which when activated opens or closes the channel, altering the
concentration of a second messenger.
 Binding of a hormone to the receptor activates the effector portion of.the receptor
directly.

Figure: General classification of “hormones” into hydrophobic and hydrophilic molecules.

Steroids are hydrophobic and act via intracellular receptors, while the hydrophilic “hormones”
act via membrane bound receptors.

Most hormonal signals are through the cAMP Second Messenger System. The signal mechanism
entails the following;

 The cAMP second messenger system is a Type 2 system which consists of a receptor,
transducer, and an effector.
 In this case, the effector is an enzyme adenylate cyclase which, when activated, converts
ATP to cyclic 3‘,5’-adenosine monophosphate (CAMP) and pyrophosphate.
 The cAMP released into the cytoplasm is the second messenger (the “hormone” is the
first messenger). The CAMP binds to its receptor in the cell, which is a regulatory
subunit of an inactive protein kinase.
 When cAMP binds, the regulatory subunit dissociates from the protein kinase.
 This protein kinase is thereby activated and it covalently modifies specific cellular
proteins by phosphorylating serine or threonine residues.

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  Phosphorylation alters the activity of these proteins, some of which are enzymes, while
others may be structural proteins (there are examples of phosphorylation causing both
activation or inactivation of proteins).
 A phosphatase is needed to remove the phosphate from these proteins and return their
activity to the basal state.

Schematic of the cAMP second messenger system

PROLACTIN:

 Prolactin (PRL) is mainly synthesized and secreted by the lactotrop cells of the pituitary.
 It is also secreted by extra-pituitary sites such as mammary gland, placenta, uterus and T
lymphocytes.
 The gene encoding PRL is unique, in humans it is located on chromosome 6, and it
contains five exons and four introns for an overall length of 10 kb, since then, an
additional exon.
 After removal of the signal peptide (28 residues), the mature form of the protein contains
199 residues (23 kDa).

The signal transduction pathway of prolactin in the female reproductive system:

 The understanding of how PRL transmits diverse signals to target cells is facilitated by
the elucidation of the JAK/Stat pathway as the prototype signaling pathway used by all
cytokine receptors.

 PRL is believed to activate sequentially the receptor by dimerizing two identical receptor
subunits, leading to activation of Jak2 kinase associated with the cytoplasmic domain.

 Hormonal stimulation of PRLR leads to tyrosine phosphorylation of several cellular

proteins, including the receptor itself. The PRLR–Jak2 interaction involves the
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 membrane-proximal region of the PRLR cytoplasmic domain, in agreement with the
ability of the short PRLR isoform to associate with the kinase.
 The activation of Janus kinases occurs by trans-phosphorylation of tyrosines upon ligand-
induced oligomerization of cytokine receptors, which brings two JAK molecules close to
each other.
 Jak2 Phosphorylates tyrosine residues on different target proteins, the best identified of
which is the receptor itself and a family of transcription factors termed signal transducers
and activators of transcription (Stats).
 These factors are signaling the molecules containing consensus domains involved in
protein–protein interactions, such as SH2 or PTB domains recruited by phosphorylated
tyrosines of the PRLR–Jak2 complex.
 Stat proteins exist within the cytoplasm in an inactive state; they are recruited by cytokine
receptor complexes through an interaction involving a phosphotyrosine (on the cytokine
receptor and/or the associated JAK) and the SH2 of the Stat protein.
 Three members of the Stat family acting as transducer molecules of the PRLR: Stat1,
Stat3 and, mainly, Stat5 (both a and b isoforms).
 Stat5 as the main mammary gland factor and is the major Stat activated by the PRLR.
 It is preferentially recruited by the C-terminal tyrosine of the receptor. Both Stat1 and
Stat3 also have been reported to be activated by the PRLR.
 After activation, Stats dimerize and migrate to the nucleus, where they specifically
interact with DNA sequences (a palindromic consensus sequence TTC xxx GAA within
the promoters of target genes.
 Then they bind to their cognate DNA-binding sequence resulting in promoter
transactivation under appropriate conditions.
 Once bound, Stats engage several elements of the transcriptional machinery, stimulating
gene expression.
 Also nuclear Jak2 regulates the amount of active nuclear transcription factor through
tyrosine phosphorylation and proteasomal degradation.
 Although the Jak2–Stat cascade is the major signaling pathway used by the PRL receptor,
other transducing pathways are also involved in signal transduction by this receptor.
 Signaling through MAP kinases (MAPK) involves the Shc/Grb2/SOS/Ras/Raf/MAPK
cascade. Activation of the MAPK pathway has been reported in different cellular systems
under PRL stimulation.
 Activation of the nucleotide exchange protein Vav has also been reported.
 PRL induces a rapid tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1)
and of the 85 kDa subunit of the phosphatidylinositol (PI)-30 kinase.
 Both PI-30 kinase and IRS-1 appear to associate with the PRLR in a PRL-dependent
manner.

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  The existence of two PRL-dependent signaling cascades has been initiated by the c-Src-
mediated activation of Fak/Erk1/2 and PI3K pathways that control the expression of c-
Myc and cyclin D1 and the proliferation.

Signal transduction pathway of PRL

In a nutshell, PRL has two cellular effects namely;

 PRL plays a critical role in corpus luteum maintenance.

 Regulates progesterone production.

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