Terminal dephosphorylation

One useful application of terminal dephosphorylation is in the isolation of terminal nucleotide sequences (Bell, Tomlinson and Tener 1964). For example, a pancreatic ribonuclease fragment containing a 3 -terminal nucleoside will be eluted from a neutral DEAE-cellulose... 

When feeding has finished, insulin secretion stops and insulin signal transduction within the cell must be terminated. Dephosphorylation of the insulin receptor by protein tyrosine phosphatase (PTPase) occurs, which inactivates the insulin receptor and insulin signalling ceases. However, there is evidence that some diabetic patients have a form of PTPase that is inappropriately active and opposes normal activation of the receptor by phosphorylation. Currently, there is a major research effort to develop drugs that inhibit PTPase and provide a new treatment for type 2 diabetes. 

Src tyrosine kinase contains both an SH2 and an SH3 domain linked to a tyrosine kinase unit with a structure similar to other protein kinases. The phosphorylated form of the kinase is inactivated by binding of a phosphoty-rosine in the C-terminal tail to its own SH2 domain. In addition the linker region between the SH2 domain and the kinase is bound in a polyproline II conformation to the SH3 domain. These interactions lock regions of the active site into a nonproductive conformation. Dephosphorylation or mutation of the C-terminal tyrosine abolishes this autoinactivation. 

Signaling by PKC is terminated by concentrations of its ligands dropping to basal levels (i.e., Ca2+ and diacylglycerol) and by dephosphorylation of the three processing sites. Dephosphorylation is controlled, in part, by a recently discovered hydrophobic phosphorylation motif phosphatase. This phosphatase, PHLPP (for PH domain Leucine-rich repeat Protein Phosphatase) dephosphorylates conventional and novel PKC isozymes, initiating their downregulation. 

Much evidence supports this scheme. For example, neuronal depolarisation increases the amount of free synapsin in the cytosol and microinjection of CAM kinase II into the terminals of the squid giant axon or brain synaptosomes increases depolarisation-evoked transmitter release. By contrast, injection of dephosphorylated synapsin I into either the squid giant axon or goldfish Mauthner neurons inhibits transmitter release. 

Figure 4.11 Dephosphorylated synapsin, associated with SSVs, is thought to form a heteromeric complex with CAM kinase II (also partially embedded in the vesicular membrane) and actin filaments. An increase in intracellular Ca + triggers phosphorylation of S3mapsin I which dissociates from the vesicular membrane. This frees the vesicles from the fibrin microfilaments and makes them available for transmitter release at the active zone of the nerve terminal...

Fludarabine is an analog of the purine adenine. It interferes with DNA polymerase to cause chain termination and inhibits transcription by its incorporation into RNA. Fludarabine is dephosphorylated rapidly and converted to 2-fluoro-Ara-AMP (2-FLAA), which enters the cells and is phosphorylated to 2-fluoro-Ara-ATP, which is cytotoxic. Fludarabine is converted rapidly to 2-FLAA. The pharmacokinetics of 2-FLAA... 

One proposed mechanism for targeting of organelles to terminals might have general implications. The synapsin family of phosphoproteins [20], which is concentrated in the presynaptic terminal, may be involved in targeting synaptic vesicles. Dephosphorylated synapsin binds tightly to both synaptic vesicles and actin microfilaments (MFs), while phosphorylation releases both of them. 

Dephosphorylated synapsin inhibits axonal transport of MBOs in isolated axoplasm, while phosphorylated synapsin at similar concentrations has no effect [21]. When a synaptic vesicle passes through a region rich in dephosphorylated synapsin, it may be cross-linked to the available MF matrix by synapsin. Such cross-linked vesicles would be removed from fast axonal transport and are effectively targeted to a synapsin- and MF-rich domain, the presynaptic terminal. 

The progression of the cell cycle is regulated by interconversion processes, in each phase, special Ser/Thr-specific protein kinases are formed, which are known as cyclin-depen-dent kinases (CDKs). This term is used because they have to bind an activator protein (cyclin) in order to become active. At each control point in the cycle, specific CDKs associate with equally phase-specific cyclins. if there are no problems (e.g., DNA damage), the CDK-cyclin complex is activated by phosphorylation and/or dephosphorylation. The activated complex in turn phosphorylates transcription factors, which finally lead to the formation of the proteins that are required in the cell cycle phase concerned (enzymes, cytoskeleton components, other CDKs, and cyclins). The activity of the CDK-cyclin complex is then terminated again by proteolytic cyclin degradation. 

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