HDGF is an abundant nuclear protein with activity as a mitogen, in that it stimulates cell cycle progression. In this study we demonstrate phosphorylation of S103 during mitosis and show this phosphorylation is required for HDGF mitogenic activity. Our study of HDGF phosphorylation in vivo was suggested by the computer search engine NetPhosK 1 [19
] 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 phosphorylation sites within a protein of interest. Separate studies had identified HDGF as a phosphorylated nuclear protein based on mass spectroscopy (MS) [16
] or by in vitro kinase assays [20
]. These studies indicated S132, S133, S165, T200 and S202 were phosphorylated in HeLa or HT-29 cells [16
]. Our results identify S103 as a new, previously unknown significant HDGF phosphorylation site not previously identified by MS. Because S103 is only phosphorylated during mitosis, based on immunostaining with a phospho-amino acid specific antibody, this likely explains why pS103 was not found by MS in non-synchronized cells. This is supported by the relatively low levels of pS103-HDGF we observed by immunoblotting whole cell extracts. It is also unclear from these global MS studies whether the peptide containing S103 was detected. We demonstrate that S165 and S202 are also phosphorylated in vivo, but at possibly lower levels in COS-7 cells relative to S103, based on differences in radiolabeling of the mutated proteins. It is of interest that previously S165 had been predicted to be a Cdk2 substrate based on sequence, however mutation of S165 had no effect on the nuclear targeting of HDGF or on its mitogenic activity [4
]. Although the kinase for S103 is not known, Salvi et al [20
] have shown that HDGF can by phosphorylated in vitro by casein kinase 2. It is not known whether S132/133 are phosphorylated 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 substitution mutation in HDGF to S103A to prevent phosphorylation nullified HDGF mitogenic activity, whereas a S103D phospho-mimic mutation was constitutively active, resulting in an increased mitogenic activity relative to wild type HDGF. This data would suggest that one model of VSMC proliferation is that activation of mitotic kinases results in phosphorylation 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 recognition sequence and requires the PWWP domain for DNA binding [21
]. It is unclear how phosphorylation regulates this process either to induce a conformational change to increase binding or enhance binding with a chromatin binding protein. The HDGF PWWP domain was recently 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 interest that a S282P mutation in the DNA methyltransferase 3b (DNMT3b, also a PWWP protein) gene results in the ICF syndrome (for immunodeficiency, centromeric instability, and facial anomalies) [22
]. 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 relation to the PWWP domain and its mutation associated with a human disease, strongly implicates these serines in the function of PWWP proteins.