A Novel Effect of MARCKS Phosphorylation by Activated PKC: The Dephosphorylation of Its Serine 25 in Chick Neuroblasts

Abstract

MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.

Synthesis and dephosphorylation of MARCKS in the late stages of megakaryocyte maturation drive proplatelet formation

“…Figure 5A reveals that despite an increase in total MARCKS (Figure 3B), the amount of P-MARCKS decreased 1.8-fold as MKs maturated and proplatelets were formed ( Figure 5A), suggesting that MARCKS phosphorylation may be detrimental to proplatelet formation. Because PMA is a potent PKC activator that leads to specific and preferential MARCKS phosphorylation, 37,38 we used it to induce MARCKS phosphorylation (schematic in Figure 5B). Treatment of MKs with PMA significantly, dose-dependently decreased the number of proplatelet-producing MKs but increased the amount of P-MARCKS ( Figure 5C).…”

“…PP2A has been shown to be the main phosphatase responsible for MARCKS dephosphorylation. 20,38,39 Therefore, we treated MKs with the specific PP2A activator C2 ceramide and PP2A inhibitor okadaic acid ( Figure 5B). Consistent with our model where MARCKS dephosphorylation is necessary for proplatelet formation, C2 ceramide significantly enhanced proplatelet formation, whereas okadaic acid significantly inhibited proplatelet formation ( Figure 5D).…”

X MARCKS the spot: myristoylated alanine-rich C kinase substrate in neuronal function and disease

“…This domain is only phosphorylated in neurons, does not affect association with membranes, and is yet to be functionally characterized (Tinoco et al, 2014 ). Interestingly, MARCKS has been demonstrated to cluster in groups with comparable phosphorylation states at the mutually exclusive S25 and ED residues (Toledo et al, 2013 ), perhaps due to the localization patterns induced by these modifications. The functional significance of phosphorylation outside of the ED requires further investigation; presently the vast majority of what is known regarding the regulation of MARCKS is in relation to phosphorylation by PKC.…”

Functional Diversification of the Four MARCKS Family Members in Zebrafish Neural Development

“…When protein sequences and their alignments are analyzed in more detail, we can see that, as it has been described for higher vertebrates alone (Li and Aderem, ; Blackshear, ), the three MARCKS conserved domains can still be recognized. Regarding the MH2, it is interesting that all nonteleost MARCKS and all teleost MARCKSA sequences share the presence of a serine phosphorylatable by Cdk5, homolog to chick S25, whose phosphorylation was previously shown to correlate to neuronal differentiation in chick and mouse embryos (Zolessi et al., ; Toledo et al., ; Contreras‐Vallejos et al., ). This serine is not present in any of the identified teleosts MARCKSB protein sequences, or in most MARCKSL1.…”

“…Of these, the MH2 domain appears as the most puzzling, as no function could yet be ascribed to it. Our group has demonstrated that it contains an extremely conserved serine residue, S25, which in the chick embryo is phosphorylated by Cdk5 in neuroblasts, some neurons, and some migrating neural crest cells, and this phosphorylation depends on the integrity of the actin cytoskeleton and on cell‐to‐cell adhesion (Zolessi and Arruti, ; Zolessi et al., ; Toledo and Arruti, ; Ruiz‐Perera et al., ; Toledo et al., ).…”