Filopodial behavior is dependent on the phosphorylation state of neuronal growth cones.
Several lines of evidence suggest that phosphorylation events play an important role in transducing neurite outgrowth signals. Here we tested if such phosphorylation events altered filopodial dynamics on neuronal growth cones and thereby might affect pathfinding decisions. The general protein kinase inhibitor K252a caused an increase in the overall length of filopodia, thereby increasing the action radius of a growth cone. Application of specific kinase inhibitors demonstrated that myosin light chain kinase, Ca/calmodulin-dependent kinase II, and protein kinase A were likely not involved in this filopodial response. Inhibition of protein kinase C (PKC) with calphostin C or cerebroside, however, induced filopodial elongation similar to that seen with K252a. Activation of PKC with the phorbol ester PMA produced the opposite effect, namely filopodial shortening. Consistent with this finding, the protein phosphatase activator C(2)-ceramide resulted in a significant increase in filopodial length, whereas application of the protein phosphatase inhibitor okadaic acid caused the opposite effect, filopodial shortening. Lastly, the tyrosine kinase inhibitor genistein also caused filopodial elongation, and this effect could be negated by the tyrosine phosphatase inhibitor sodium ortho-vanadate. Using the calcium indicator fura-2, we further showed that these drugs did not cause a measurable change in the free intracellular calcium concentration ([Ca(2+)](i)) in growth cones. Taken together, these results suggest that the action radius of a growth cone and its resulting pathfinding abilities could be rapidly altered by contact with extracellular cues, leading to changes in the activity of protein kinases and phosphatases.
Cheng
S
S
Biology Department, Georgia State University, Atlanta, GA 30303, USA.
Mao
J
J
Rehder
V
V
D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D002554:Cerebrosides / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D016257:Fura-2 / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D020439:Growth Cones / Q000378:metabolism*; D026121:Indole Alkaloids; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011554:Pseudopodia / Q000502:physiology*; D015398:Signal Transduction; D012908:Snails; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D002227:Carbazoles; D002352:Carrier Proteins; D002554:Cerebrosides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D026121:Indole Alkaloids; D047908:Intracellular Signaling Peptides and Proteins; C064769:N-acetylsphingosine; D009281:Naphthalenes; C015482:protein kinase modulator; D019319:Okadaic Acid; D014638:Vanadates; C049985:staurosporine aglycone; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D017027:Protein Tyrosine Phosphatases; C058819:calphostin C; D013110:Sphingosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2