for HDGF mitogenic activity. Our study of H DGF phos-
phorylation in vivo was suggested by the computer
search engine NetPhosK 1  that matches amino acid
sequence to known protein kinase phosphorylation
motifs, with statistical ranking for significance. This type
of search engine is useful for identifying potential phos-
phorylation sites within a protein of interest. Separate
studies had identified HDGF as a phosphorylated
nuclear protein based on mass spectroscopy (MS)
[16,17] or by in vitro kinase assays . These studies
indicated S132, S133, S165, T200 and S202 were phos-
phorylated in HeLa or HT-29 cells [16,17]. Our results
identify S103 as a new, previously unknown significant
HDGF phosphorylation site not previously identified by
MS. Because S103 is only phosphorylated during mito-
sis, based on immunostaining with a phospho-amino
acid specific antibody, this likely explains why pS103
was not found by MS in non-s ynchronized cells. This i s
supported by the relatively low levels of p S103-HDGF
we observed by immunoblotting whole cell extracts. It is
also unclear from these globa l MS studies whether the
peptide containing S103 was detected. We demonstrate
that S165 and S202 are also phosph orylated in vivo, but
at possibly lower level s in COS-7 cells relative to S103,
based on differences in radio labeling of the mutated
predicted to be a Cdk2 substrate based on sequence,
howev er mutation of S165 had no effect on the nuclear
targeting of HDGF or on its mitogenic activity [4,5].
Although the kinase for S103 is not known, Salvi et al
 have shown that HDGF can by phosphorylat ed in
vitro by casein kinase 2. It is not known whether S132/
133 are phosphoryl ated in vivo or whether S132/133
phosphorylation is functionally significant.
We found that phosphorylation of HDGF-S103 has a
significant effect on HDGF mitogenic activity. A substi-
tution mutation in HDGF to S103A to prevent phos-
phorylation nullified HDGF mitogenic activity, whereas
a S103D phospho-mimic mutation was constitutively
active, resulting in an increased mitogenic activity rela-
tive to wild type HDGF. This data would suggest that
one model of VSMC proliferation is t hat activation of
mitotic kinases results in p hosphorylation of S103-
HDGF, leading to increased cell proliferation. As the
impact of the S103 mutants on the cell cycle was much
more profound than the wild type protein, this would
suggest that HDGF mitogenic function is dependent on
phosphorylation and not just dependent on the amount
of HDGF present.
Although the mechanism of phospho-S103-HDGF
function during mitosis is unclear, it is of interest that
another HDGF family member LEDGF, demonstrates
metaphase chromatin binding, requiring cooperative
interaction of the PWWP and AT-hook domains.
Although HDGF does not contain AT-hook domains, it
does bind DNA directly requiring a large 36 bp recogni-
tion sequence and requires the PWWP domain for
DNA bindin g . It is unclear how phosphorylation
regulates this process either to induce a conformational
change to increase binding or enhance binding with a
chromatin binding prot ein. The HDGF PWWP domain
was recent ly shown to dimerize on heparin and whether
phosphorylation plays a role in potentially regulating
HDGF dimerization on chromatin via the PWWP
domain is an area of active research.
It is of great in terest that a S282P mutation in the
DNA m ethyltransferase 3b (DNMT3b, also a PWWP
protein) gene results in the ICF syndrome (for immuno-
def iciency, centromeric instability, and facial anomalies)
. This serine is 4 amino acids carboxy to the PWWP
domain in DNMT3b, and homologous to the location of
S103 in HDGF. The conservation of this serine in rela-
tion to the PWWP domain and its mutation associated
with a human disea se, strongly implicates these s erines
in the function of PWWP proteins.
HDGF is a mitotic phosphoprotein and phosphorylation
of S103 plays an important ro le in regulating the prolif-
eration of cells and the mitogenic function of HDGF.
Department of Pediatrics, Cardiology Division, Johns Hopkins University,
600 N. Wolfe Street, Baltimore, 21287, USA.
Center for Cell Signaling and
Department of Microbiology, University of Virginia, 1400 Jefferson Park
Avenue, Charlottesville, 22908, USA.
ADE conceived the experiments and wrote the manuscript. JY made the
phospho HDGF mutants, generated the in vitro phosphorylation data and
drafted that experimental section. MR performed nocodazole and cell
sorting experiments and drafted the experimental results. PD performed cell
transfections and immunohistochemical analyses. DLB edited the draft and
contributed significantly to experimental design. All authors have read and
approved the final manuscript.
Received: 6 July 2009 Accepted: 13 April 2011 Published: 13 April 2011
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