We continue to investigate further the molecular basis of topographic phosphorylation of cytoskeletal proteins using squid giant axon system. To understand the mechanisms whereby compartment-specific metabolic patterns are regulated in the highly asymmetric neuron with its distinct soma, axonal and dendritic compartments, the ideal system is the giant fiber system in the squid. Here, for biochemical studies, it is possible to separate pure axoplasm from giant axons from the large cell bodies in the giant fiber lobe from which they originate. This cannot be done in the much smaller mammalian neurons. For several years our laboratory, using the squid giant fiber system, has focussed on the kinases responsible for the phosphorylation of neurofilament proteins and has shown dramatic differences in the level and pattern of phosphorylation in the two compartments. Recently we have turned to the phosphatases, that also play a key role in regulation. Accordingly, our efforts each summer are to: (a) use Western and ICC analyses to identify the compartment-specific kinases/phosphatases involved using specific antibodies;(b) study the cross-talk interactions between kinases and phosphatases and (c) explore the factors that regulate the activities of these enzymes in each cellular compartment. Among these is the role of Pin1, a peptidyl-prolyl cis/trans isomerase that isomerizes phospho Ser/thr-proline residues of which there are many in squid neurofilaments. In addition to these studies we are investigating the development of the squid nervous system so as to (1) study the development of the IIIrd order giant fibers in the stellate ganglion;(2) establish the neuroanatomical substrate in which to analyze the expression of neuronal-specific genes (in collaboration with Dr. Peter Burbach, Utrecht), and (3) to explore the developmental expression of neuronal regulatory peptide FMRF-amide, recently cloned from the squid stellate ganglion . In addition we continue to study the neuron specific kinase Cdk5, which in the squid hatchling, shows extensive expression in the mantle musculature, suggesting a role in muscle development. As we have done in previous years, our biochemical studies compare whole giant axons (or extruded axoplasm) and the giant fiber lobe (GFL) which contains the cell bodies that, by axonal fusion, generate the giant axons. In previous studies we have established the following;(1) Axoplasm extracts display much higher levels of protein phosphorylation than GFL extracts with many more proteins serving as substrates. (2) Axonal neurofilament proteins, particularly the high molecular weigh NF220, are highly phosphorylated at multiple KSP repeat sites in the tail domain. On the other hand, NFs from cell bodies are not phosphorylated. (3) Cell bodies and axons share the same active kinases, NF sub unit proteins and regulators yet phosphorylation of the tail domain is restricted to the axonal compartment. The obvious question is why, and of course, how? (4) P13 chromatography of cell body and axoplasm lysates (a procedure that extracts Cdc2-like kinases and associated proteins) revealed that the axonal multimeric complex of proteins was significantly more active than the cell body complex, although both, for the most part, shared similar kinases and substrates. (4) GFL extracts express higher levels of tyrosine phosphatase activity than axoplasm extracts suggesting that this difference may, in part, account for the different patterns of protein phosphorylation in each compartment. To determine the role of tyrosine phosphatase activity we are studying """"""""cross-talk"""""""" regulation of the MAP kinase pathway by tyrosine kinases/phosphatases. To test this, we are comparing the level of MAP kinase activity in GFL and axoplasm extracts since Erk1/2, a key kinase in the pathway, is known to phosphorylate KSP repeats in NFs. The hypothesis predicts that higher tyrosine phosphatase levels in the GFL should down-regulate the Erk1/2 pathway. Meanwhile, we continue to explore by Western blotting and ICC assays, the expression of other kinases (PKA and PKC) as well as tyrosine and ser/thr phosphatases in GFL and axoplasm, to identify the """"""""players"""""""" that may be involved in each compartment. Since the antibodies used are primarily those prepared from mammalian antigens, the results are difficult to interpret unless proper negative controls (pre-incubation of antibodies with specific blocking peptides) are used to eliminate the possibility of cross reactivity with non-specific squid epitopes. In subsequent studies we detected a dramatic difference in the phosphorylation of MARCKS protein, an actin binding protein that is regulated by PKC phosphorylation. GFL lysate phosphorylation of MARCKS substrate was considerably greater than that in axoplasm, which is virtually inactive. We also demonstrated a significant cross-talk interaction between PKA and PKC activities. This suggests an important regulatory role for PKC and PKA in the GFL and we plan to explore this further by (1) comparing PKC and PKA activities in each compartment using various substrates (2) comparing the level of expression of PKC isomers in Western blots and ICC of stellate ganglia (3) Incubating GFL in PKC and PKA inhibitors and activators to determine their effects on protein phosphorylation in each compartment. These studies will provide a better understanding of topographic neuronal cytoskeletal protein phosphorylation.

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Boumil, Edward F; Vohnoutka, Rishel; Lee, Sangmook et al. (2018) Assembly and turnover of neurofilaments in growing axonal neurites. Biol Open 7:
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