Biomedical Engineering Reference
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secretion. Thus there are several observations which provide additional
support for the suggestion that the MARCKS family of proteins play a
role in integrating Ca
2+
and PKC-dependent signals in the regulation of
neurosecretion.
Recent studies (Ohmitsu
et al.,
1999;Yamauchi
et al.,
1998) suggest that
MARCKS is also phosphorylated by proline-directed protein kinase such
as microtubule-associated protein (MAP) kinase and cycline-dependent
protein kinase (Cdk5). A mass spectroscopic analysis of intact MARCKS
purified from bovine brain revealed at least 6 phosphorylation sites in the
N-terminal domain which were upstream of the phosphorylation sites for
PKC (Taniguchi
et al.,
1994). In addition Yamauchi
et al.
97b; 1998 found
additional phosphorylation sites in the C-terminal region of rat MARCKS.
Thus Ser residues in both the N-terminal and C-terminal regions (Ser
291
and
Ser
299
in rat MARCKS
)
have been shown to be followed immediately
by prolines. More recently Ohmitsu
et al.
(1999) reported that glutamate
acting at the NMDA receptor induced a long lasting phosphorylation of
MARCKS in primary cultures of rat hippocampal neurones. Unexpectedly
this phosphorylation was not inhibited by the PKC inhibitor calphostin or
by down regulation of PKC with phorbol esters but was inhibited by the
MAP kinase kinase inhibitor PD 098059. This study also found that gluta-
mate induced a rapid, transient phosphorylation of MARCKS which was
inhibited by calphostin. Of particular interest in the study of Ohmitsu
et al.
(1999) is their observation that whereas PKC induced phosphoryla-
tion of MARCKS prevented the binding of calmodulin to MARCKS MAP
kinase induced phosphorylation had little effect on the interaction between
calmodulin and MARCKS. In contrast the introduction of phosphates into
MARCKS by MAP kinase prevented the interaction between MARCKS
and F-actin to the same extent as PKC induced phosphorylations. Thus
PKC is not the only kinase that phosphorylates MARCKS which casts
doubt on the use of MARCKS as a specific marker for PKC activation
when cells are stimulated with various ligands. However as Schonwasser
et al.
(1996) reported the Ser 113 immediately adjacent to proline in
murine MARCKS was not phosphorylated by TPA or platelet derived
growth factor in Swiss 3T3 cells the ability of MAP kinase to phosphory-
late MARCKS may be limited to only certain cell types. It remains to
be determined whether MARCKS in SH-SY5Y cells can be phosphory-
lated by MAP kinase. Of interest also are reports (Schmitz
et al.,
1998) that
poly ADP-ribose (formed in parallel to DNA strand breaks) binds strongly
to the MARCKS family of proteins and inhibits PKC induced phosphory-
lation of and calmodulin binding to MARCKS. Thus MARCKS proteins
and actin could be targets of the poly (ADP-ribose) DNA damage signal
pathway.
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