MAP Kinase Signaling

Cells exist in a very dynamic environment, not only on the inside of the cell membrane, but also on the outside.  Thus, cells must be able to interact with their outside environment and respond to stimuli appropriately.  One of the several signaling pathways involved in communication from the outside of the cell inward is the (very general) MAP kinase (MAPK) signaling pathway.  The cell uses the MAPK signaling pathway for several reasons, primarily to amplify signals.  Additionally, aberrant MAPK signaling is implicated in several types of cancers.

First, MAPK signifies mitogen activated protein kinase - a fancy word for a protein that responds to mitogens, or a molecule that stimulates mitosis.  Proteins of the MAPK family were discovered in 1989 in yeast, with ERK1 being the first mammalian MAPK signaling protein, involved in insulin signaling pathways (at the time at least...).

The MAPK signaling pathway is often called a cascade because the components in the pathway amplify a signal within the cell.  The three major components are:

• MAPKKK / MEKK / MAP3K: The MAP kinase kinase kinase.  This protein phosphorylates MAP kinase kinase.  Several forms are found within the cell and their abundance is lower than that of MAPKK or MAPK; thus, MAPKKK isn't as involved in the amplification of signals.

• MAPKK / MEK / MAP2K: The MAP kinase kinase.  MAPKK phosphorylates MAPK and is highly abundant within the cell, but its only substrate is MAPK.  Just a few phosphorylation events on MAPKK result in significant activation of MAPK.

• MAPK / ERK: The MAP kinase.  MAPK is also abundant in the cell and has diverse substrates, including itself.

There are several pathways that involve MAPK signaling.  Two of the important ones are:

1. The ERK pathway: growth factor stimulation of cell surface receptors (via receptor tyrosine kinases [RTKs]) causes activation of Ras, which activates Raf (MAPKKK) to activate MEK (MAPKK) and then ERK (MAPK).  ERK's several targets include Elk and Ets, which control cellular proliferation.
2. The JNK pathway: Stress on the cell results in activation of several proteins, including Rho, which goes on to activate MEKK, then MEK, and JNK.  JNK's major targets include c-Jun, ATF2, and Elk1, which control proliferation and apoptosis.

One important consideration is the specificity of MAPK signaling.  Specificity can be achieved in several manners: protein components can only "fit" other specific protein components.  Additionally, the spacial organization of a cell can affect specificity, and this type of specificity is often controlled by scaffold proteins.  

MAPK signaling is essential for many cellular processes, including proliferation, apoptosis, embryonic development, and cancer progression.  Many intricacies of the pathways are still being worked out and will provide significant insight in the future.

The Basics of Protein Translation

Proteins, a major constituent of the cell, have many diverse functions and are classically considered to be the "work horses" of the cell.  Additional molecules, such as RNAs and lipids, have shown importance in signaling and catalyzing chemical reactions; however, proteins remain an important part in the life of a cell.

Before proteins can perform their evolved functions, they must be synthesized within the cell.  The process of synthesizing a protein is critically important to the cell and, thus, is an energy-intensive process.  

The basic building blocks of a protein are amino acids, which come in twenty (and more) flavors.  These amino acids have different properties that afford proteins different structures and functions when the amino acids are polymerized together in distinct orders.  These amino acids can have distinct signaling roles when they exist as monomers as well (see this paper by Nobukuni et al for an example).  

Monomeric amino acids in the cellular environment do not randomly polymerize to form proteins.  In the first of several steps, tRNAs are charged: they are covalently linked to amino acids via aminoacyl tRNA synthetases.  These synthetases hold the very important role of attaching the appropriate amino acid to the appropriate tRNA.  Because inappropriate charging of tRNAs would lead to misincorporation of amino acids into a protein chain (wasting energy or leading to even bigger problems for the cell), sythetases are very specific.  In fact, some synthetases have an editing site, where they will catalyze the removal of incorrectly placed amino acids.

After tRNAs are charged with their appropriate amino acids, they are ready for interaction with the ribosome.  Ribosomes are large, complex molecules that merit their own post.  Briefly, ribosomes are composed of RNA and protein and are made of two distinct complexes: the large and small subunits (depending on the origin of the ribosome, the subunits have different sedimentation coefficients, so you might see 30S and 50S for bacteria or 60S and 40S for eukaryotes, for example).  Ribosomes catalyze the polymerization of amino acids into proteins.

In the first step of ribosome-mediated protein production, the small subunit of the ribosome combined with a tRNA for methionine (the amino acid that begins the protein chain) scans along the transcribed mRNA until it encounters a start site (ATG codon).  Here, the complex stops, and the charged tRNA with its amino acid comes into contact with the peptidyl transferase site (P) on the ribosome.  eIF2 (eukaryotic initiation factor 2), which was along for the ride, hydrolyzes GTP to GDP at this point such that the ribosome stops at the appropriate codon.

Next, the large subunit of the ribosome binds the small subunit, making a full ribosome that is ready for catalysis.

The tRNA that lines up with the mRNA's next codon then binds in the acceptor site (A), along with the help of eEF-1 (eukaryotic elongation factor-1), which hydrolyzes GTP to GDP.  At this point, the ribosome goes into action: using the peptidyl transferase center (PTC), it catalyzes the covalent linkage of the first and second amino acids.

The entire ribosome now moves along the mRNA in a process called translocation, which requires the help of EF-2 (and GTP hydrolysis).  The first tRNA is moved into the exit site (E), and the second tRNA moves into the P site, while the A site is open for another aminoacyl-tRNA.

The process repeats until the ribosome encounters a stop codon.  At this point, termination factors (TFs), which have structures similar to tRNAs and bind mRNAs but do not have amino acids, enter the acceptor site of the ribosome.  The ribosome then catalyzes the addition of water to the end of the amino acid chain, releasing it from the peptidyl transferase center and allowing it to leave the ribosome and begin folding into its native conformation.

While there are several details that I may have seemingly glazed over, this post should give a broad, simplified overview of translation.  Future posts will address the many details involved.

The Many Functions of Ubiquitin

Ubiquitin (abbreviated as Ub) is described as a small, frequently-encountered molecule involved in the proteosomal degradation of proteins.  In reality, the addition of ubiquitin moieties to proteins can serve several functions and is now recognized as a common post-translational modification (PTM).

The classical ubiquitin pathway that first comes to mind is that of proteosomal degradation.  In this pathway, illustrated in the accompanying figure, ubiquitin is first "activated" by binding covalently to a cysteine residue on E1, the ubiquitin-activating enzyme, which requires the energy of ATP.  Then ubiquitin is transfered to a cysteine residue on E2, the ubiquitin-conjugating enzyme (no ATP is required here).  Finally, E2 binds to a scaffold called E3, the ubiquitin ligase, which also contains the target protein - illustrated in pink.  This target protein is then covalently linked to ubiquitin via a lysine residue.  Several rounds of this pathway then result in linear polyubiquitination of proteins, which is then recognized by other components in the cell that result in its recruitment to the proteasome and subsequent degradation (a topic of future posts).  The end result is that the target protein is broken down into small seven-to-eight amino acid pieces.

As mentioned above, there are three proteins involved in the attachment of ubiquitin to target proteins, E1, E2, and E3.  In the cell, E1s are the least plentiful (only one has been discovered thus far), while the human genome codes for tens of E2.  Several more E3 ligases have been discovered (and more every day).

Importantly, ubiquitination does not exclusively function in proteosome-mediated degradation of proteins.  Monoubiquitination, or the attachment of a single ubiquitin moiety to a protein, has been shown to have diverse effects on target proteins (see listbelow). Additionally, multiple monoubiquitination events can occur on a single protein, as can branched polyubiquitination.

Ubiquitin is not the sole small protein modifier.  Several molecules similar to ubiquitin have been described and exhibit diverse functions in cellular signaling (topics of future posts).  These proteins include SUMO (small ubiquitin-like modifier), NEDD, FATIO, and FUBI.  Needless to say, all of these proteins have some of the most adorable names in science.

The following list is a short compilation of ubiquitin functions, and, as we all know very well, no hard-and-fast rules exist for ubiquitin modifications (or science in general).

• p53 ubiquitination by MDM2: cell cycle regulation, apoptosis
• HIF1 ubiquitination by VHL: hypoxic response
• Caspase 3 and 7 ubiquitination by XIAP: apoptosis
• PCNA: DNA repair
• RNA polymerase II: transcriptional regulation
• H2AX and H2A: transcriptional regulation, protein recruitment

Of course, there are more proteins that are modified by ubiquitin, but I hope that the above table illustrates the diversity of the modification.

Interestingly, ubiquitin modifications are not forever: de-ubiquitinating enzymes (DUBs) remove ubiquitin moieties from tagged proteins.  Thus, the reversible ubiquination represents a dynamic cellular process.

Some cool (and important) papers and links:
Principles of ubiquitin and SUMO modifications in DNA repair
Non-traditional Functions of Ubiquitin and Ubiquitin-binding Proteins
The Ubiquitin System
Nonproteolytic Functions of Ubiquitin in Cell Signaling
Ubiquitin Function and Variety

Initiation of Eukaryotic Transcription IV

Changing Up the Chromatin

All of the factors that affect transcription that have been described are involved in changing the ability of RNA polymerase and transcription factors to bind to and initiate DNA transcription.   An additional method of modulating transcription is via changes to the chromatin (see writings on that here).  Histones and other proteins are able to change the packaging of DNA and, thereby, affect transcription.  The compaction of the DNA directly affects the accessibility of it to RNA polymerase and transcription factors.  Euchromatin is the active form of chromatin that is not fully compacted; in contrast heterochromatin is generally not active or accessible.  The presence of histone H1 (the linker histone, and, let’s be honest, everyone’s favorite histone) also affects the compaction of the chromatin.

How exactly do histones affect transcription?  Put simply, they prevent other proteins from binding the DNA, especially the DNA that is facing the histone core.  Further, DNA is wrapped around histones, which changes the structure of the DNA.  Such distortions can affect binding sites and preclude transcription factor binding.  If the transcription factors and RNA polymerase are to bind to the DNA, the chromatin must be unraveled and the DNA must become accessible.  One way to do this is to move the histones out of the way, opening up binding sites.  Histones are incredibly dynamic and move around on the DNA frequently, wrapping and unwrapping different sequences.  With the fluctuations of the chromatin, transcription factors can bind while the histones “breathe.”

An additional mechanism to allow access to the DNA is via histone modifications on the N-terminal domains.  The N-termini of the histones contain lysine groups that affect DNA-histone interactions.  Therefore, affecting the charged residues via acetylation or methylation, for example, will change the interactions between the histones and DNA.  Acetylation of a histone tail effectively neutralizes its charge such that its structure is altered and no longer binds DNA as tightly.  Modifications on histones can also form binding sites for transcription sites.  Chromodomains bind to methylated lysine residues, while bromodomains bind acetylated lysines.  There are a number of different modifications that affect transcription, and new effects are still being elucidated.

-          Acetylation of lysine / arginine: transcription induction

-          Phosphorylation of serine, threonine, or tyrosine: transcription induction; chromatin compaction

-          Methylation of arginine: transcription induction

-          Methylation of lysine: gene silencing

-          Ubiquitination of H2A and H2B: degradation; transcription; growth regulation

-          ADP-ribosylation: histone repelled from DNA at sites of repair

Activation of Transcription Initiation III

Activation Domains

Activation domains are another important portion of the regulatory protein that is involved in altering the activity of a promoter.  There are three main types of activation domains:

1.      Acidic, such as Gal4

2.      Glutamine-rich, such as Sp1

3.      Proline-rich, such as CTF

These different types of activation domains have different mechanisms and may also be involved in allowing the regulatory elements to function at a distance.  Importantly, many regulatory proteins may have multiple activation domains. 

As mentioned previously, the mediator complex works to integrate the signals from multiple activation domains and passes this signal along to RNAPII.  There are about 20 different subunits that bind to RNAPII and different activation domains. 

To determine the functional domains of an activator, we can use reporter genes.  Ideally, we would cotransfect a plasmid containing the protein of interest and a plasmid containing a reporter  (such as lacZ) that is transcribed only when the activation domain of the protein of interest is transfected.  In this way, we can examine different regions of proteins to determine the precise domains that are involved in activating transcription. 

An additional way to determine where an activation domain is in a protein is to use a Gal4 hybrid assay.  This method involves using a domain swap, which uses the DNA-binding domain of Gal4 and other domains from the protein of interest.  By measuring the activity of a reporter gene, such as lacZ, we can determine if the domain that is bound to Gal4 is an important activation domain. 

Co-activators

First, we have activators that are recruited to genes, which are involved in regulating transcription and also bind the DNA directly.  Co-activators, in contrast, are recruited to the promoter but do not bind DNA.  They form complexes and can assemble on the DNA-binding proteins.  In this way, co-activators can interact with proteins essential for transcription, such as the machinery, histone modifiers, and chromatin-remodeling complexes.  Important to note is that some co-activators, such as VP16, CBP, and GCN5, have acetyltransferase activity. 

VP16 is a herpesvirus protein that contains an acidic activation domain and interacts with host cell factor (HCF).  When VP16 binds HCT and OCT1, which is a DNA-binding activator but no activation domain, it promotes the assembly of the PIC and helps to initiate transcription by targeting TBP, TFIIB, and TAF40.

GATA4 is another important co-activator that is a zinc finger DNA-binding protein that is involved in heart development.  It works via the recruitment of TBX-5.

Architectural Factors that Affect Transcription

The main way that architectural factors affect transcription is via DNA bending.  These proteins do not have a transactivation domain, as do other regulatory factors, but they do affect the interactions between activators, co-activators, and the PIC.  This is often accomplished by bending the DNA and shortening the distance between cis-acting elements.  Such bending of the DNA allows for transcriptional regulators to act at a distance. 

The HMG proteins are small, abundant proteins that function to change the DNA architecture.  These proteins do not have high sequence specificity and can bind the minor groove to induce a bend in the DNA.  Bending of the DNA facilitates complex assembly and nucleosome remodeling, which may change the rate of transcription.