Phosphorylation mechanism and structure of serine-arginine protein kinases

Abstract

The splicing of mRNA requires a group of essential factors known as SR proteins, which participate in the maturation of the spliceosome. These proteins contain one or two RNA recognition motifs and a C-terminal domain rich in Arg-Ser repeats (RS domain). SR proteins are phosphorylated at numerous serines in the RS domain by the SR-specific protein kinase (SRPK) family of protein kinases. RS domain phosphorylation is necessary for entry of SR proteins into the nucleus, and may also play important roles in alternative splicing, mRNA export, and other processing events. Although SR proteins are polyphosphorylated in vivo, the mechanism underlying this complex reaction has only been recently elucidated. Human alternative splicing factor [serine/arginine-rich splicing factor 1 (SRSF1)], a prototype for the SR protein family, is regiospecifically phosphorylated by SRPK1, a post-translational modification that controls cytoplasmic-nuclear localization. SRPK1 binds SRSF1 with unusually high affinity, and rapidly modifies about 10-12 serines in the N-terminal region of the RS domain (RS1), using a mechanism that incorporates sequential, C-terminal to N-terminal phosphorylation and several processive steps. SRPK1 employs a highly dynamic feeding mechanism for RS domain phosphorylation in which the N-terminal portion of RS1 is initially bound to a docking groove in the large lobe of the kinase domain. Upon subsequent rounds of phosphorylation, this N-terminal segment translocates into the active site, and a β-strand in RNA recognition motif 2 unfolds and occupies the docking groove. These studies indicate that efficient regiospecific phosphorylation of SRSF1 is the result of a contoured binding cavity in SRPK1, a lengthy Arg-Ser repetitive segment in the RS domain, and a highly directional processing mechanism.

Figure 1. SR Protein Domain Structure

All traditional SR proteins have one or two N-terminal RRMs and one C-terminal RS domain. The amino acid sequence for the RS domain of the prototype SR protein SRSF1 is displayed. Peptide mapping and cellular analyses indicate that the phosphorylation of two segments in this RS domain (RS1 & RS2) by SRPK and CLK kinases control subcellular distribution.

Figure 2. Structural Features of SRPK1

A) Ribbon diagram of SRPK1 in complex with ADP and a short peptide substrate (RRRERSPTR). The peptide binds near the MAP kinase (MAPK) insert. SRPK1 lacks most of its N-terminus (1–41) and insert domain (256–473). B) Several conserved structural elements and contacts in SRPK1.

Figure 3. Mechanism of SRSF1 Phosphorylation by SRPK1

A) Dual Track Mechanism. Start-trap analyses indicate that SRPK1 can phosphorylate up to 8 serines in RS1 using a processive mechanism in which the kinase stays attached to substrate after each round of phosphorylation. The remaining serines in RS1 are modified in a distributive manner in which the kinase and substrate dissociate after each phosphorylation event. B) Directional phosphorylation. Mapping studies show that SRPK1 is a directional kinase that initially binds to an initiation box (Ser221–225) in the center of the RS domain and then moves in an N-terminal direction to maximally phosphorylate RS1. The bold and light arrows indicate that processivity is progressively diminished as SRPK1 translates from the C-to-N-terminus and dissociation becomes favored over forward cataysis.

Figure 4. Model Describing How the RS domain of SRSF1 Is Threaded Into the Active Site of SRPK1

A) X-Ray Structure of the SRPK1:SRSF1 Complex. SRSF1 retained the central RRM2 and RS1 segments and lacked RRM1 and RS2. Only a portion of RS1 is well defined in the complex (N’-RS1, residues 204–210) and resides in the electronegative docking groove. The dotted circles present the possible path of the segment of SRSF1 disordered in the crystal from N’-RS1 to RRM2 and the active site. The surface rendition of CLK1 is shown in the right panel. The dotted circles represent a possible path of the p-RS1 peptide substrate on the kinase. B) Feeding Mechanism. The N-terminal portion of RS1 (N’-RS1) initially binds in the docking groove while the C-terminal portion (initiation box) occupies the active site. Representative Arg-Ser pairs in both segments are represented as green hexagons. The dotted line represents intervening Arg-Ser-rich regions in RS1. In the presence of ATP, RS1 is phosphorylated in a C-to-N direction until residues 191–198 (β4 of RRM2) occupies the docking groove. Electropositive side chains from the P+2 pocket stabilize the phosphates on RS1.

Figure 5. Surface Electrostatic Properties of SRPK and CLK Kinases

Ribbon (top) and electrostatic surface presentations (bottom) for Sky1p, SRPK1 and CLK1 are displayed. All three molecules were crystallized as truncated proteins. The non-conserved N-terminal and spacer domains were deleted in Sky1p and SRPK1. The N-terminal RS domain was deleted in CLK1.