14 -3 -3 is an abundant, predominantly phospho-binding protein, intimately
involved in the regulation of many diverse signal transduction events including cell
cycle regulation, nucleo-cytoplasmic targeting of essential transcription factors and
regulation of catecholamine synthesis. The 14 -3 -3 family consists of 7 isoforms
(denoted a, ß, y, 8, c, rl and in mammals and shows a degree of isoform specificity
in binding target proteins. 14 -3 -3 is phosphorylated in an isoform specific manner, for
example SDK1 /PKD phosphorylates 14 -3 -3 11,13 and (, but not cs and T. Our
laboratory has previously identified in vivo 14 -3 -3 phosphorylation sites, S185 and
S233. Phosphorylation of S233 by the serine /threonine protein kinase Casein kinase
la (CK1 a) was shown to negatively affect the interaction with Raf kinase. The group
of Gotoh have recently shown that phosphorylation of S185 by the stress activated
kinase c -Jun NH2- terminal kinase (JNK) negatively effects the interaction with Bax.
During studies in our laboratory that identified CKla as a 14 -3 -3 kinase, several other
proteins co- purified through four steps of chromatography, including centaurin -a1 and
CPI -17, suggesting a protein complex - these interactions have subsequently been
characterised. CK1 has a potential phosphorylation dependent 14 -3 -3 binding site
within the same region previously shown to be the interaction site for centaurin -al
and the aim of this investigation was to examine the possible interaction.
14 -3 -3 was found to associate with CK1 isoforms, with CKIa being studied
further to reveal an interaction through serine residues 218 and 242 in a
phosphorylation dependent manner, in vivo. Centaurin -a1 was found to interact with a
region corresponding to residues 214 -226, only if S218 was in a dephosphorylated
state, suggesting a possible regulatory mechanism. Mutagenesis of CKIa suggests
that S242 is a high affinity binding site for 14 -3 -3, with S218 being of lower affinity.
Investigations to identify possible kinase(s) responsible for phosphorylation of CK1
showed that stimulation of PKA can increase CKla:14 -3 -3 association in cells, but
PKA does not appear to phosphorylate CKIa in vitro.
As phosphorylation of 14 -3 -3 itself is an important regulatory mechanism,
attempts were made to produce antibodies to phosphorylated S185 and S233 on 14 -3-
3. A phospho -specific antibody to S185 was successful, but antibodies to a- phospho-
S233 had no preference to the phosphorylation state of 14 -3 -3, although they were of
high selectivity and affinity for 14 -3 -3 isoforms.
The kinase BCR (Breakpoint cluster region) is an important, but poorly
understood protein that has been shown to associate with and phosphorylate 14 -3 -3.
Investigations showed that BCR phosphorylates 14 -3 -3 on Ser233 in vitro.
Additionally, BCR is shown to associate with another two isoforms of 14 -3 -3 (e and
rl) both in vitro and in vivo. However 14 -3 -3 a did not associate with BCR in vitro.
BCR selectively phosphorylates 14 -3 -3 r more than , in contrast to CK1.
In summary, Interactions with CK1 and 14 -3 -3 are characterised in detail and
a possible regulatory mechanism discussed for CK1:centaurin- a1:14 -3 -3 interaction.
Further insights into BCR signalling are revealed by identification of the
phosphorylation site on 14 -3 -3 that has been shown to negatively affect binding to
Raf kinase.
