Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament‐Associated Kinase and by Casein Kinase I

Abstract

We have shown previously that a neurofilament (NF)‐associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF‐M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI‐7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two‐dimensional phosphopeptide maps of NF‐M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross‐reactivity of NFAK with an antibody raised against CKI. We have also identified Ser502, Ser528, and Ser536 as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser528 and Ser536 and flanking amino acids are highly conserved in higher vertebrate NF‐M sequences. Neither Ser502 nor Ser536 has been identified previously as NF‐M phosphorylation sites.

Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain

“…2C), respectively. The missing spots V2, V3, V8, and L2 are relatively poorly labeled in vivo and were not visible in these autoradiographs (Hollander et al, 1993(Hollander et al, , 1996Bennett et a!., 1994).…”

“…Recently, we reported the sequences of severa! peptide fragments that are phosphory!ated in vivo and are also phosphory!ated in vitro by both casein kinase I and an NF-associated kinase (Hollander et a!., 1996). These peptides are a!!…”

“…They were separated by high-voltage electrophoresis (1,000 V for 35-60 mm) at pH 4.7, followed by chromatography in a buffer consisting of isobutyric acid/pyridine/acetic acid/n-butanol/water (65:5:3:2:29, by volume) (Hollander et a!., 1993(Hollander et a!., , 1996Bennett et a!., 1994). The locations of phosphopeptides were revealed by autoradiography.…”

“…% TFA. Eluates were concentrated and passed over Sephadex GlO spin columns to reduce the low molecular weight UV-absorbing contaminants derived from the cellulose (Hollander et al, 1996).…”

“…These include casein kinase I Hollander et al, 1996) and at least three different Pro-dependent kinases: protein kinase FA/glycogen synthase kinase-3 (GSK3; Guan et al, 1991), cyclin-dependent kinases (Hisanaga et al, 1991;Guan et al, 1992;Lew et a!., 1992;Shetty et al, 1993;Sun et a!., 1996), and mitogenactivated protein kinase (ERK kinase; Roder and Ingram, 1991;Chertoff et al, 1995). The phosphorylation sites are mostly in Ser residues in Lys-Ser-Pro (KSP) sequences (Geisler et a!., 1987;Lee et a!., 1988), which are repeated as many as 60 times in mammalian NF-H. Other, less frequently repeated, potential phosphorylation motifs have also been recognized, especially in NF-M, including KXSP and KXXSP (Shaw, 1991).…”