At a recent CLL meeting in Copenhagen Daniel Catovsky, one of the greatest hematologists of his generation, was heard to applaud a paper that actually showed pictures of CLL cells while he muttered something derogatory about pictures of gels. There were murmurs of approval throughout the hall. As hematologists our primary skill was to be able to recognize blood cells down the microscope. Increasingly hematology papers are not about that, but show pictures of bands on gels that are unfamiliar to most of us. In order to understand these papers some basic understanding is required.
So a visit to Wikipedia is in order.
Phosphorylation is the addition of a phosphate (PO4) group to a protein molecule or a small molecule. Reversible phosphorylation of proteins is an important regulatory mechanism that occurs in cells. Enzymes called kinases (phosphorylation) and phosphatases (dephosphorylation) are involved in this process. Many enzymes and receptors are switched "on" or "off" by phosphorylation and dephosphorylation. Reversible phosphorylation results in a conformational change in the structure in many enzymes and receptors, causing them to become activated or deactivated. Phosphorylation usually occurs on serine, threonine, and tyrosine residues.
One such example of the regulatory role that phosphorylation plays is the p53 tumor suppressor protein. The p53 protein is heavily regulated and contains more than 18 different phosphorylation sites. Activation of p53 can lead to cell cycle arrest, which can be reversed under some circumstances, or apoptotic cell death. This activity occurs only in situations wherein the cell is damaged or physiology is disturbed in normal healthy individuals.
Upon the deactivating signal, the protein becomes dephosphorylated again and stops working. This is the mechanism in many forms of signal transduction, for example the way in which antigen stimulates a B lymphocytes.
Elucidating complex signaling pathway phosphorylation events can be difficult. In a cellular signaling pathways, a protein A phosphorylates protein B, and B phosphorylates C. However, in another signaling pathway, protein D phosphorylates A, or phosphorylates protein C. Global approaches such as phosphoproteomics, the study of phosphorylated proteins, which is a sub-branch of proteomics combined with mass spectrometry-based proteomics, have been utilised to identify and quantify dynamic changes in phosphorylated proteins over time. These techniques are becoming increasingly important for the systematic analysis of complex phosphorylation networks.
There are thousands of distinct phosphorylation sites in a given cell since: 1) There are thousands of different kinds of proteins in a lymphocyte. 2) It is estimated that 1/10th to 1/2 of proteins are phosphorylated. 3) Phosphorylation often occurs on multiple distinct sites on a given protein.
Since phosphorylation of any site on a given protein can change the function or localization of that protein, understanding the "state" of a cell requires knowing the phosphorylation state of its proteins. For example, if amino acid Serine-473 ("S473") in the protein AKT is phosphorylated, AKT is, in general, functionally active as a kinase. If not, it is an inactive kinase. (For explanation of Akt - see previous entry).
Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine is the most common, followed by threonine. Tyrosine phosphorylation is relatively rare. However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies, tyrosine phosphorylation sites are relatively well understood.
Antibodies can be used as powerful tools to detect whether a protein is phosphorylated at a particular site. Antibodies bind to and detect phosphorylation-induced conformational changes in the protein. Such antibodies are called phospho-specific antibodies; hundreds of such antibodies are now available. They are becoming critical reagents both for basic research and for clinical diagnosis. Phosphorylation replaces neutral hydroxyl groups on serines, threonines, or tyrosines with negatively-charged phosphates. Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges. The relative amount of each isoform can also easily and rapidly be determined from staining intensity on 2D gels.
Having got that out of the way, let’s turn to a paper in this month’s Blood from Marta Muzio and colleagues from Federico Caligaris Cappio’s laboratory in Turin, entitled Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy.
There seems to be very good evidence that stimulation of the lymphocyte via the B-cell receptor (BCR) is very important in the pathogenesis of CLL. That stimulation triggers the phosphorylation of syk which then passes the phosphate parcel down a number of possible pathways. One of the difficulties in understanding all this is the stange names given to these enzymes. Syk stands for spleen tyrosine kinase, Y being the single letter symbol for the amino acid Tyrosine that is used by biochemists. When you come across these acronyms it is worth Googling them to find out what they stand for.
Five or six years ago we showed that only about 50% of CLLs would respond to BCR stimulation by phosphorylating syk. We expected that these would be the ones with unmutated VH genes, but although there was an association it was by no means precise. Fecerico Caligaris Cappio has always had the idea that CLL cells were anergic cells, unable to react to normal stimuli beause their signaling mechanism has been dulled by constant low grade stimulation by autoantigens – it is one of the mechanisms that stops us from continually attacking our own tissue.
In mice anergic B cells have a characteristic molecular signature, showing constitutive expression (ie switched on all the time) of tyrosine phosphorylation with activation of the MAPK ERK that cannot be further induced after BCR stimulation.
(Explanation: MAPK, mitogen activated protein kinase. First named MAP kinase in 1988, but from one of its specific substrates: microtubule associated protein (MAP-2). By 1989, it was realized that this was the same as the 42 kDa protein, phosphorylated by mitogen stimulation, known since 1981, and so it was retro-acronymically renamed mitogen activated protein kinase. When the protein was cloned in 1990 it was named, not MAPK, but ERK1 for extracellular-signal regulated kinase, a somewhat uninspired choice. MAPK is now more commonly used for the superfamily of related kinases of which the ERK family is one.)
Muzio et al in this paper show that the cells from CLL patients who are unable to respond to BCR stimulation also have constitutive phosphorylation of ERK without evidence of Akt phosphorylation. The only anomaly in this paper is that there seems to be no correlation with prognostic factors.
Thus this paper is added evidence to support Federico’s long held hypothesis.